The procedures you will perform today have two main objectives To prepare media for selective growth of E.coli cells containing ‘cloning’ plasmids To observe a human cancer cell line growing in tissue culture media and prepare an aliquot of human cancer cells for RNA extraction

Week 1 Day 1: Preparation of Bacterial growth media and Tissue Culture of human cells

The procedures you will perform today have two main objectives
To prepare media for selective growth of E.coli cells containing ‘cloning’ plasmids
To observe a human cancer cell line growing in tissue culture media and prepare an aliquot of human cancer cells for RNA extraction

You will perform the following procedures
Prepare LB antibiotic selection agar plates
• You will use these plates in the transformation experiment you perform on in the ligation and transformation of E.coli experiment day.
• The plates will allow you to ‘select’ for E.coli cells that contain plasmids with an antibiotic resistance gene
Prepare LB antibiotic selection broth (liquid) media
• You will use this media for selective growth of antibiotic resistance plasmid containing E.coli cells, in liquid culture.
Inoculate a colony of plasmid containing E.coli cells into liquid selection media
• You will then ‘grow’ the E.coli culture overnight in order to have sufficient plasmid containing cells for plasmid extraction on day 2
Observe the phenotype and determine the cell number and viability of cells of a Chronic Myeloid Leukaemia (CML) cell line
• You will use a microscope to observe and count CML cell line growing in a standard growth medium
• You will use an ‘automated cell counter’, to count and determine the viability of CML cells
• You will prepare and ’store’ an aliquot of these cells for RNA extraction on week 1 day 3

Preparation of LB antibiotic selection agar plates
Video of how to prepare the agar plates (note this video shows the protocol up to stage 5).
1. Add 8g of Miller’s LB agar media into the 250mL Duran bottle labelled Agar
2. Add distilled water up to the 200mL mark and gently swirl to mix
3. Place a small piece of autoclave tape on the top of the bottle lid and label the tape with your name and the date
4. Autoclave the media at 121°C for 15 minutes (this will sterilize the media)
5. Cool the media and store at room temperature
6. On week 2 day 1, you will ‘melt’ the media in a microwave, cool again and add antibiotic

Preparation of LB antibiotic selection broth (liquid) media
Note: One bottle of LB ampicillin broth media will be made per group of 4 students.
1. Add 2.5g of tryptone, 1.25g of yeast extract and 2.5g of NaCl (sodium chloride) into a Duran bottle.
2. Add distilled water to the 250mL mark
3. Place a small piece of autoclave tape on the top of the bottle lid and label the tape with your name and the date
4. Autoclave the media at 121°C for 15 minutes (to sterilize the media)
5. Cool the media to a temperature of about 50°C
6. Under sterile conditions, transfer 5mL of the broth into a 15mL Falcon tube. Label the tube with your name/ID number and the date
7. Add 2.5μL of 50mg/mL kanamycin stock solution to achieve a final concentration of 25μg/mL*
8. Keep the bottle on your desk until required

*Stock solution concentration
Example calculation for a 25μg/mL final concentration in final 50mL solution, from a 50mg/mL starting concentration.
Calculation C1 x V1 = C2 x V2 (C1 = stock starting concentration, C2 = desired final concentration. V1 = volume to add of stock, V2 = desired final volume of media).
C1= 50mg/mL, C2 = 25μg/mL, V1 =?, V2 = 5mL. Rearrange equation to calculate V1:
V1 = (C2 x V2)/C1 = (25μg/mL x 5mL)/50,000μg/mL
= 0.0025mL = 2.5μL

Observe and record the phenotypes of cells
You will examine the human cancer cell line growing under standard ’tissue culture’ conditions. You will record phenotype observations (using an inverted light microscope), and determine the cell concentration (per ml), and cell viability (the % ‘alive’) and size distribution of the cells using an automated cell counter – the BioRad (TC20)

Counting mammalian tissue culture cells using the BioRad automatic cell counter (TC20) and prepare an aliquot of human cancer cells for RNA extraction
Preparing cells for automated counting
1. Dispense 10μL of 0.4% Trypan blue solution into the lid of a 1.5mL microfuge tube.
2. Ensure the cell suspension is well mixed and ‘de-clumped’ by pipetting the cell suspension up and down at least 3-4 times, then transfer approximately 1mL of cell suspension into the 1.5mL microfuge tube
3. Using a p10 and fresh 10μL filter tip, place the tip into the cell suspension in the bottom of the 1.5 mL tube and gently pipette up and down 3-4 times. This will ‘pre-wet’ the tip and aid in pipetting accuracy and final counting.
4. Next pipette 10μL of cell suspension into the 10μL of Trypan blue solution tube in the lid of the 1.5mL microfuge tube.
5. Mix the cell suspension into the Trypan blue by gently pipetting up and down 3-4 times, then pipette 10μL of the Trypan blue/cell suspension into one of the slots of the BioRad counting slide
6. Insert the TC20 counting slide into the counter chamber of the TC20. The count should start automatically.
7. Record the total cell number and viable cell number you obtain in your lab book notes.
8. The TC20 data file will be saved and the data file uploaded to Class Results
TC10 cell counting.ppt
565.0 KB

Video on calculating cell numbers using an haemocytomter
How to prepare a standard reusable haemocytometer slide and how to estimate cell numbers using an haemocytometer slide
Use a 70% ethanol/water solution to wipe the haemocytometer slide base clean, then dry the slide totally.
1. Moisten the shoulders of the haemocytometer (you can do this by breathing on the slide) and affix the coverslip using gentle pressure and small circular motions. The phenomenon of Newton’s rings can be observed when the coverslip is correctly affixed. This correct fixing ensures the depth of the chamber is correct.
2. Proceed to apply the cell sample for counting as outlined above.

Watch this video for an overview of cell counting of mammalian cultured cells
Counting mammalian tissue culture cells using the BioRad automatic cell counter (TC20) and prepare an aliquot of human cancer cells for RNA extraction

Preparing an aliquot of human cancer cells for RNA extraction
1. Remove all the cell/Trypan blue solution from the lid of the 1.5ml microfuge tube
2. Place the tube into the centrifuge, making sure the tube hinge is facing ‘outwards’ and spin the cell suspension at 450g (4000rpm) for 5 minutes
3. Aspirate off the old media, taking care not to dislodge the cell pellet.
4. Add 100μL of RNAprotect Cell Reagent and store the cells at -20°C until required

Inoculation of E.coli for plasmid amplification
1. Transfer approximately 5mL of liquid LB antibiotic broth media into 15 mL Falcon tube
2. Take a sterile 1μL inoculation loop and pick a single large blue E.coli colony from a selection plate and inoculate it into the LB/antibiotic media in the Falcon tube
3. Transfer the tubes to a 37°C rotary incubator set to a rotation of 250rpm. The culture will be ‘grown’ overnight until it reaches an optical density (OD) of approximately 0.3 OD.
4. You will use this culture on week 1 Day 2 to extract and purify the plasmid

Week 1 Day 2: Preparation of plasmid from E.coli and restriction digest cloning

The procedures you perform here have one final objective.
To purify sufficient linearised phosphatase treated plasmid for used in the ‘cloning’ligation and transformation of E.coli

You will perform the following procedures.
Purify plasmid DNA from a culture of E.coli grown in selection broth.
• You will use a commercial ‘plasmid purification’ spin column kit to purify the plasmid from the E.coli culture you inoculated on Day 1
Determine the concentration of the purified plasmid DNA
• You will use the Qubit Flourometer to determine the plasmid concentration in your preparation.
• You use this figure to determine how many μLs (containing a certain number of ng) of purified plasmid solution you will use in the restriction enzyme digest reactions
Perform a restriction enzyme digest and phosphatase treatment on an aliquot of purified plasmid
• Firstly you will use a restriction enzyme to digest (cut) the circular plasmid into linear plasmid
• As a group you will then run a gel to analyse your undigested (control) and digestedplasmid DNA

Purifying plasmid DNA from a culture of E.coli grown in selection broth. Using Sigma GenElute PLN350 plasmid purification kit.

• Insert a GenElute miniprep Binding Column into a 2 mL tube, dispense 500 μL of Column Preparation Solution into the top of the binding column and centrifuge at 13,000rpm for 1 minute
• Discard the flow-through liquid into your liquid waste collection flask, keep the GenElute column and microcentrifuge tube
• Centrifuge your ‘overnight’ E.coli culture at 4000 rpm for 4 minutes. Decant the liquid into your waste flask and use a pipette to aspirate of any remaining liquid. Be careful doing this NOT to remove your cell pellet
• Dispense 200 μL of Resuspension Solution (stored at 4°C) into the cell pellet. Fully resuspend the pellet with no visible ‘lumps’ by carefully pipetting up and down 2-3 times.
• Dispense 200 μL of Lysis Solution to the cell pellet and ‘invert’ the tube 6 times to mix and Incubate for exactly 4 minutes until the mixture becomes clear and viscous. Do not vortex.
• Dispense 350 μL of Neutralisation Solution into tube and mix thoroughly by inverting the tube at least 6 times. Do not vortex.
• Centrifuge at 13,000rpm for 10 minutes.
• Taking care not to transfer any of the ‘white precipitate’ pour the mixture into the centre of the GenElute column and centrifuge at 13,000rpm for 1 minute.
• Discard the flow-through from the collection tube into your waste flask, keep the GenElute column and microcentrifuge tube
• Dispense 750 μL of Wash solution into the centre of the GenElute column and centrifuge at 13,000rpm for 30 seconds
• Discard the flow-through from the collection tube into your waste flask, keep the GenElute column and microcentrifuge tube
• Centrifuge at 13,000 rpm for 1 minute
• Keep the GenElute column. Discard the flow-through into your liquid waste flask and the collection tube into your solid waste collection bag.
• Label a clean 2 mL collection tube and place the GenElute column into the top of the labelled tube
• Pipette 50 μL of Elution Solution into the centre of the GenElute column and centrifuge at 13,000rpm for 1 minute
• The liquid in the collection tube (elute) should contain your purified plasmid DNA. Keep the labelled collection tube in your rack until ready to perform Qubit analysis.

Determining the concentration of purified plasmid DNA
Using the Invitrogen Qubit flourometer. Use these video link guides 1 and 2 for information on how to carry out the protocol

Protocol
• Prepare 600 μL of working reagent, this will be sufficient to carry out 3 estimations
• Dispense 3μL of Quant-IT dsDNA BR dye and into a sterile 1.5 mL microfuge tube. Dispense 600 μL of Quant-IT dsDNA BR buffer solution into the tube and vortex for 10 seconds.
• Label 3 Quant-IT reaction tubes: 1, 2 (these will be the standards) and S (the plasmid DNA sample). Write labels on the tops of the tubes not on the sides.
• Prepare tubes 1 and 2 standards as follows
• Dispensing 190 μL of the prepared Quant-IT working solution into tube 1 and add 10 μL of standard 1
• Dispensing 190 μL of the prepared Quant-IT working solution into tube 2 and add 10 μL of standard 2
• For your sample analysis, dispense 5μLs of DNA into tube S and 195 μL of Quant-IT working solution
• vortex all the tubes for 2-3 seconds and then incubate for 2-5 minutes at room temperature.
• Set the Qubit to determine DNA Standard concentration values
• Use standards 1 and 2 to calibrate and then obtain the reading for your sample
• Record your values in your lab book and the Class Results Excel file.

Perform a restriction enzyme digest and phosphatase treatment on an aliquot of purified plasmid. In this experiment you will digest a a sample of your purified plasmid using a restriction enzyme. You will prepare one digestion reaction and one control reaction. NOTE: At some stage during an incubation, your group should prepare a 150 mL – 0.8% agarose/0.5X TAE gel Introduction and Class Results

Setting up the control reaction
• Label a clean 1.5 mL microfuge tube
• Dispense XμL (containing approximately 100ng) of purified plasmid DNA sample into the tube
• Dispense YμL of ddH2O into the tube to bring the volume up to 18μL
• Dispense 2μL of x10 restriction enzyme buffer into the tube
• Flick the tube to mix then flash spin to collect all the reagents to the bottom of the tube

Setting up the restriction digestion reaction
• Label a clean 1.5 mL microfuge tube
• Dispense XμL (containing approximately 500ng) of your purified plasmid sample into the tube
• Dispense YμL of ddH2O to the tube to make the volume up to 17μL
• Dispense 2μL of x10 restriction enzyme buffer into the tube
• Bring the tube to a demonstrator and add 10 enzyme units each of restriction enzymes to your tube (PstI and SgfI – buffer C)
• Flick the tube to mix then flash spin to collect all the reagents to the bottom of the tube

Incubate both reactions for the following temperatures and times
• Place both tubes in the 37°C heating block for 1hr minimum (2hrs, in this case is better).This digestion should enable the restriction enzymes to ‘cut’ the circular plasmid DNA molecules into linear DNA molecules. The longer the time more of the plasmid will be digested, until it is ‘fully’ digested.
• After incubation, record how long you digested your DNA sample
• Remove the control reaction from the heating block and allow it to cool to room temperature
• Remove 4μL of the restriction digest reaction from it’s tube and add to a fresh 1.5mL tube. Return the digestion reaction to the 37°C heating block

Prepare an agarose gel to analyse uncut and digested treated plasmid
As a group, prepare a 150mL 0.8% agarose gel.

Loading the gel
• Dispense 5μL of x5 DNA loading dye into the control reaction tube and ‘mix’ by pipetting the liquid up and down 2 – 3 times
• Dispense 1μL of x5 DNA loading dye into the digestion reaction aliquot tube and mix by pipetting the liquid up and down 2 – 3 times
• Collect any drops of sample that may have adhered to the tube sides or lid to the bottom of the tube by ‘flash-spinning’ in a centrifuge
• Load samples into the gel in the order indicated below
Lane 1: 5μL of a DNA ladder (Promega 1KB ladder)
Lane 2: all 25μL the control reaction.
Lane 3: all 5μL of the digestion reaction.
Lane 4: leave this lane empty
• Run the gel for a minimum of 45mins at 150v

Week 1 Day 3: Purification of plasmid from agarose gel and preparation of human cancer cells for RNA extraction

The procedures performed on this day have 2 objectives
To gel purify and quantify linear digested/phosphatase treated plasmid
To observe and record the phenotypes of human cancer cell line after growth in media containing a TKI ‘drug’ and to prepare TKI treated cells for RNA extraction

You will perform the following procedures.
Purification of linearised plasmid from an Agarose gel
• You will use an agarose gel to separate the fragment(s) of your restriction digested reaction
• You will then use a commercial gel extraction/spin column purification kit to purify the digested plasmid
To observe and record the phenotypes of cells and prepare for RNA extraction
• You will determine the total cell number and viability of a CML cell line that has been growing for 72hrs in media supplemented with a ‘TKI’
• You will then prepare an aliquot of these TKI treated cells for later extraction of total RNA

As a group, prepare a 150mL 0.8% agarose gel to purify digested plasmid.
NOTE: you will use SybrSafe as the DNA staining agent

Load samples into the gel in the following order
Lane 1: 5μL of a DNA ladder.
Lane 2: all the aliquot of the digestion reaction
Once all the samples have been loaded, run the gel at 50-100 volts for approximately 30 minutes, or until separated. We will observe how far the DNA has migrated in the gel using ‘blue light’. Once sufficient separation of fragments has occurred i.e. the DNA we wish to purify can be clearly identified, based on the estimation of its predicted molecular weight, you will be able to proceed to the gel extraction protocol.

Sigma GenElute Gel Extraction kit
Video protocol http://www.sigmaaldrich.com/video/biology/na1111.html
• Label and weigh a sterile 1.5 mL tube (record the weight, write it on the side of the tube)
• Excise the band from the gel using the x-tracta Gel Extraction Tool* and dispense it into the 1.5 mL tube
• Add 3 gel volumes* of Gel solubilzation Buffer into the tube (*if slice weighs 100mg use 300 μL of buffer)
• Incubate the tube at 60°C for 10 minutes or until the slice is fully dissolved (vortex the tube every 2-3 minutes to ensure sufficient mixing/heating).
• Take a GenElute Column G filter column* and place it into a 2 mL collection tube *MAKE SURE YOU USE THE CORRECT COLUMN
• Dispense 500 μL of Column preparation solution into the top of the column.
• Centrifuge at 13,000rpm (12,000g) for 1 min
• Discard the eluate from the collection tube, keep the GenElute G column
• From step 4, check that the gel slice has fully dissolved into the Gel solubilzatin Buffer and that the colour of the gel mixture is still yellow (similar to fresh Gel Solubilization Solution with no gel slice). If it is proceed to the following step. If the color of the mixture is red, show a demonstrator
• Dispense 1 gel volume of 100% isopropanol into the tube and mix by pipetting until homogenous.
• Dispense all of the solution of into the top of the GenElute Column G filter column
• Centrifuge at 13,000rpm for 1 min
• Discard the flow-through from the collection tube into your waste bottle, keep the GenElute column
• Dispense 700 μL of Wash Solution G into the centre of the column
• Centrifuge at 13,000rpm for 1 min
• Discard the flow-through from the collection tube into your waste bottle, keep the GenElute column and replace it into the collection tube
• Centrifuge at 13,000rpm for 1 min
• Discard the flow-through from the collection tube into your waste bottle and the collection tube into a solid waste collection bag, keep the GenElute column G
• Place the GenElute column into the top of a clean labeled 2 mL collection tube.
• Pipette 50 μL of pre-heated Elution Solution* into the centre of the GenElute column (*incubate the elution solution to 65°C before use).
• Centrifuge at 13,000rpm for 1 min
• Keep the collection tube (the eluate should contain the purified DNA).
• Discard the GenElute column into a solid waste collection bag.
• Store the purified DNA at 4°C, in a labelled tube!

Record phenotypes of cells growing in ’TKI’ media
Sometime during the day – groups will observe human cancer cell growing in TKI media and then prepare an aliquot of the cells for extraction of total RNA. Using the light microscope examine the cells in the TC flask and compare the phenotyes of cells to those you observed of the control cells. Have the cells changed in morphology, if so how and why? What percentage of cells look ‘different’? What do you think has happened to the cells due to the ‘activity’ of TKI drug?

Prepare a flask of human cancer cells for RNA extraction
Watch this video for an overview of cell counting of mammalian cultured cells
Counting mammalian tissue culture cells using the BioRad automatic cell counter (TC20) and prepare an aliquot of human cancer cells for RNA extraction

Preparing cells for automated counting
• Dispense 10 μL of 0.4% Trypan blue solution into the lid of a 1.5mL microfuge tube.
• Ensure the cell suspension is well mixed and ‘de-clumped’ by pipetting the cell suspension up and down at least 3-4 times, then transfer approximately 1mL of cell suspension into the 1.5mL microfuge tube
• Using a p10 and fresh 10μL filter tip, place the tip into the cell suspension in the bottom of the 1.5 mL tube and gently pipette up and down 3-4 times. This will ‘pre-wet’ the tip and aid in pipetting accuracy and final counting.
• Next pipette 10μL of cell suspension into the 10μL of Trypan blue solution tube in the lid of the 1.5mL microfuge tube.
• Mix the cell suspension into the Trypan blue by gently pipetting up and down 3-4 times, then pipette 10 μL of the Trypan blue/cell suspension into one of the slots of the BioRad counting slide
• Insert the TC20 counting slide into the counter chamber of the TC20. The count will automatically start.
• Record the total cell number and viable cell number you obtain in your lab book notes.
• The TC20 data file will be saved and the data file uploaded to Class Results

Preparing an aliquot of human cancer cells for RNA extraction
• Remove all the cell/Trypan blue solution from the lid of the 1.5ml microfuge tube
• Place the tube into the centrifuge, making sure the tube hinge is facing ‘outwards’ and spin the cell suspension at 450g (4000rpm) for 5 minutes
• Aspirate off the old media, taking care not to dislodge the cell pellet.
• Add 100μL of RNAprotect Cell Reagent and store the cells at -20°C until required

Counting mammalian tissue culture cells: see Week 1 Day 1: Preparation of Bacterial growth media and Tissue Culture of human cells

he procedures you perform on this day one final objective
To prepare RNA samples from cells that contain mRNA of sufficient concentration and quality for use in cDNA synthesis

You will perform the following procedures.
Purify total RNA from cultured human cells
• You will use a commercial spin column purification kit to purify the total RNA from the cells you prepared in Week 1
Use a standard denaturing agarose gel electrophoresis to determine the quality of an RNA sample
• You will work in groups to prepare a denaturing agarose gel.
• You will use this gel to determine the quality of your RNA by assaying the 28s – 18s ratio of your RNA samples.
Use the BioRad Experion system to determine RNA quality and concentration
• You will observe how to prepare RNA samples for BioRad Experion microfluidic chip analysis
• This analysis determines the ‘quality’ of your RNA sample as an RQI value (RNA Quality Indicator) – an estimated value of how ‘intact’ the RNA is in a sample. This value is again determined by the 28s – 18s ratio of the RNA sample. You will compare this reading to you gel images
• This system will also determine the concentration of the sample to be used in an RT-PCR reaction

Purify total RNA from cultured human cells
Important general notes for working with RNA
RNases are very stable enzymes that have the ability to degrade RNA. Autoclaving solutions and glassware is insufficient to remove or deactivate RNases. Therefore the first step when preparing to work with RNA is to create an RNase-free environment. The following precautions are recommended as a best defense against ‘losing’ your RNA due to RNAse activity.
• The area used for your RNA extraction should be located away from microbiological work stations
• Clean all surfaces with commercially available RNase decontamination solutions (e.g. RNA-Zap)
• Clean, disposable gloves should be worn at all times when handling reagents, samples, pipettes, disposable tubes, etc. It is recommended that gloves are changed frequently (and/or ‘sprayed’ e.g. RNA-Zap, or similar agents) to avoid RNAse contamination
• Use designated tips, tubes, pipettes, etc specifically prepared for RNA only work
• All solutions for RNA work should be prepared using at least 0.05% DEPC-treated autoclaved water or molecular biology grade nuclease-free water
• When working with purified RNA samples, ensure that they remain on ice during downstream applications or they will degrade.

The experiment
In this stage of the experiment we will extract RNA from the cells you prepared on Week 1 Day 3. For the extraction method we are using it is recommend that a starting number of no more than 1 x 10^7 cells per extraction is used, so as not to ‘overload’ the purification column. For this experiment the required number of cells have been pre-determined (BioRad TC counter figures) and prepared in RNAprotect Cell Reagent. Note: Immediate stabilization of RNA in sorted or cultured cells is generally a prerequisite for reliable gene expression analysis using microarray, real-time RT-PCR, or other nucleic acid-based technology. This is because changes in the gene expression pattern occur immediately after harvesting due to unspecific and specific RNA degradation as well as to transcriptional induction. RNA stabilisation can be achieved by using the RNAprotect Cell Reagent.

Purification of RNA using the Qiagen RNAeasy kit
The procedure provides an enrichment for mRNA, since about 15-20% of total RNA is comprised of RNAs of <200 nucleotides in length, including 5.8S rRNA, 5S rRNA, and tRNAs, are excluded from purification. Perform all the steps at room temperature (15–25°C). For optimum results, try to work as quickly and efficiently as possible. • Centrifuge the tube containing the cells in RNAprotect, for 2 min at (10000 rpm) • Use a pipette to carefully remove and discard the supernatant, a couple of μl can be left to ensure you keep the cell pellet. • Add Xμl RLT lysis solution* to the cell pellet and vortex for 15 seconds. *For < 5 x 10^6 use 350μl of Buffer RLT. For 5 x10^6 –1x10^7 cells use 600μl. • Pipette all the lysate into a QIAshredder homogeniser column and centrifuging for 2 minutes at 14,000rpm. • Transfer the homogenized lysate to a gDNA Eliminator spin column placed in a 2 ml collection tube. Centrifuge for 30s at 14000 rpm. • Examine the column to make sure that no liquid remains on the column membrane after centrifugation. If necessary, repeat the centrifugation until all liquid has passed through the membrane. Once you are satisfied remove and discard the column, keep the flow-through • Measure how much flow-through liquid you have and an equal volume of 70% ethanol to it. Mix well by pipetting up and down • Place an RNA binding column into a collection tube and transfer 700μl of the flow through/ethanol solution into the top of the column. • Centrifuge for 15s at 14,000 rpm. Discard the flow-throughand replace the column back into the collection tube • Add 700 μl of RW1 Buffer to the centre of the spin column, and centrifuge for 15 s minutes at 13,000 rpm. Discard the flow-through and replace the column back into the collection tube • Add 500 μl of RPE Buffer to the centre of the spin column, and centrifuge for 15 s at 13,000 rpm. Discard the flow-throughand replace the column back into the collection tube • Add 500 μl of RPE Buffer to the centre of the spin column, and centrifuge for 2 minute at 13,000 rpm. Discard the flow-throughand replace the column back into the collection tube • After the final wash remove the spin column from the collection tube, discard the flow-through, open the lid of the column, replace the column into a clean collection tube and centrifuge for 2 minute at 13,000 rpm. • Place spin column into a new RNase free 1.5 ml collection tube, add 30 μl of elution solution (RNAse free water) directly to the centre of the spin column membrane, close the lid and centrifuge the column for 1 min at 14,000rpm. • Measure the quantity of RNA using the Qubit fluorometer Determining the concentration of purified RNA sample using the Invitrogen Qubit flourometer. Use these video link guides 1 and 2 for information on how to carry out the protocol Protocol • Prepare of working reagent, sufficient to carry out 3 estimations • Dispense 3μL of Quant-IT RNA BR dye and into a sterile 1.5 mL microfuge tube, add 600 μL of Quant-IT RNA BR buffer solution into the tube and vortex for 10 seconds. • Label 3 Quant-IT reaction tubes: 1, 2 (these will be the standards) and S (the RNA sample). Write labels on the tops of the tubes not on the sides. • Prepare tubes 1 and 2 standards as follows • Dispensing 190 μL of the prepared Quant-IT working solution into tube 1 and add 10 μL of standard 1 • Dispensing 190 μL of the prepared Quant-IT working solution into tube 2 and add 10 μL of standard 2 • For your sample analysis, dispense 5μLs of RNA sample into tube S and 195 μL of Quant-IT working solution • vortex all the tubes for 2-3 seconds and then incubate for 2-5 minutes at room temperature. • Set the Qubit to determine RNA BR standard concentration values • Use standards 1 and 2 to calibrate and then obtain the reading for your sample • Record your values in your lab book and the Class Results Excel file. Introduction and Class Results Use denaturing agarose gel electrophoresis to determine RNA quality Preparing a denaturing agarose gel for RNA analysis. You will perform this analysis in groups of 4 using a 50mL volume mini gel. • Weigh out 0.3 g of ultra-grade agarose into a DEPC treated Duran bottle. • Add 27 mL of ultra-pure water to the bottle, replace the lid, ensuring that the lid of the bottle is loose, to avoid buildup of pressure, • Heat the mixture in the microwave, until the agarose is dissolved, swirling the vessel occasionally to ensure uniform mixing. Be careful not to let the agarose solution boil over. If the volume of liquid reduces considerably during heating, due to evaporation, add more ultra-pure DEPC treated water to the bring the volume back to the original 27 mL. This will ensure that the agarose concentration is correct. • After heating, cool the agarose slurry, swirling occasionally to prevent uneven cooling, until the temperature reaches approximately 65°C, then add 3mL of x10 Formaldehyde-Free RNA Gel Solution. This solution contains buffering agents, and an alternative denaturing agent to formaldehyde. • Pour the agarose solution into an assembled gel casting tray, immediately insert the comb and let the gel set. • Place the gel in the electrophoresis tank and fill the tank with enough x1 Formaldehyde-Free RNA running Solution to cover the gel to approximately 1mm above the level of the gel. The 1mm depth level can be 'estimated' by inserting a clean 10 μL pipette tip into the buffer until it just touches the surface of the gel and visualising the depth level. • Once satisfied with the buffer level, carefully remove the comb from the gel. • Let the gel equilibrate in the running buffer for at least 30 min before loading and running the gel. Preparing RNA samples for analysis using denaturing agarose electrophoresis RNA must be denatured before running on an agarose gel. To denature the RNA, a specific RNA loading buffer is added to each RNA sample and then the sample is heated and cooled before and loading onto the gel. • Add XμL (containing approximately 100ng) of your RNA sample into a sterile DEPC treated 1.5 mL tube • Add YμL of RNase free water into the tube to bring the volume up to 8μL • Add 8μL of RNA loading buffer* into the tube • Add 3μL RNA ladder and 2μL of RNA loading buffer* into a tube • Incubate the sample and ladder for 10 min at 70°C. This will denature the RNA. • Place samples on ice for at least 5 minutes then load the gel • Load your samples into the RNA gel in the following order Lane 1: All 5μL of the RNA ladder solution Lane 2: All 16μL of the RNA sample solution *The RNA loading buffer serves four main purposes: 1. To denature the RNA sample prior to loading. 2. To increase the density of the samples to ensure that they sink into the wells on loading (this is the same as for DNA gels). 3. To add coloured running dye and an RNA visualisation dye to the samples and facilitating loading 4. To add an RNA visualisation dye so the GelDocEZ imager can image the RNA in the gel Promega RNA ladder The BioRad Experion microfluidic system for determination of RNA concentration and quality. Preparation of RNA sample for analysis on a ‘standard sensitivity’ Experion RNA microfluidic chip • Pipet 2μl of the RNA sample into an RNase-free PCR tube. • Denature by heating at 70°C for 2 min and then immediately place on ice for 5 min. • Flash spin load sample onto the chip • Remember the well number you are given and record this in your lab book notes • The separation procedure will be performed for you and you will be given the results 3. BioRad Experion microfluidic system for determination of RNA concentration and quality Watch this video for an overview of the importance of RNA quality for cDNA synthesis and qPCR analysis Week 1 Day 5: Synthesis and testing of cDNA The procedures you perform on this day one final objective To prepare ‘quality checked’ cDNA to be used for PCR amplification of our transcripts of interest An overview of RT-PCR (cDNA) synthesis You will perform the following procedures. Create cDNA from quality confirmed total RNA • You will use the BioRad iScript Advanced cDNA synthesis kit and a PCR thermal cycler to create cDNA from your quality confirmed total RNA sample. This kit uses a combination of Oligo (dT)20 and random hexamer primers to amplify all the RNA transcripts present in your RNA sample, into single stranded cDNA. Determine the quality of cDNA for PCR amplification • You will perform a set of real-time qPCR reactions to check the 'efficiency and uniformity’ of the cDNA synthesis reaction, and detect the presence of any ‘contaminating’ genomic DNA (gDNA) in your cDNA Create cDNA from quality confirmed total RNA • Add YμLs of an RNA template (containing 1μg RNA) and XμLs of qPCR grade water to the tube, to bring the volume up to 19μL. Refer to the class data file Introduction and Class Results • Bring your tube, on ice, to the demonstrator and you will then add 4μLs of iScript advance qPCR buffer and 1μL of iScript advanced reverse transcriptase enzyme • Return the tube back to the ice and keep the tube on ice until the class is ready to load the thermal cycler and run the cDNA synthesis program Running the cDNA synthesis reactions As a class we will then load the thermal cycler and run the cDNA synthesis program using the parameters below. This will synthesis the cDNA Once your cDNA reaction is complete, add 180μL of qPCR grade water to the cDNA reaction and store on ice until ready to ‘test’ your cDNA Determining the quality of cDNA for PCR amplification PrimePCR Controls • Collect a set of 4 white qPCR tubes and one green 1.5mL tube from the demonstrator. • The white qPCR tube set contains, in order 1. qPCR tube containing exactly 1μL of TROVE2 qPCR primer set 2. qPCR tube containing exactly 1μL of TROVE2 5’ASS primer set 3. qPCR tube containing exactly 1μL of TROVE2 WT exon 2 splice site primer set 4. qPCR tube containing exactly 1μL of ‘contaminating’ genomic DNA (gDNA) detection primers • The green 1,5mL tube contains exactly 40μL of BioRad Sso Advanced SYBR green qPCR mix • Add 40μL of your diluted cDNA to the green tube, mix well, flash spin and then transfer exactly 19μL of the mix into each of the white qPCR tubes • Bring the tubes, on ice, to the demonstrator and we will then load the Biorad CFX connect real-time qPCR thermal cycler • We will then run the qPCR and perform the analysis using the Biorad CFX manager software Week 2 Day 1: PCR amplification of transcripts of interest The procedures you perform on this day have one final objective To PCR amplify specific transcripts present in the cDNA samples and digest them ‘ready’ for ligation You will perform the following procedures. Use PCR to amplify Actin from cDNA • The purpose of this procedure is to 'test' the cDNA for PCR amplification of gene of interest. • You will use β-actin specific PCR primers to amplify any Actin transcripts that were amplified into cDNA in the RT-PCR reaction performed on Week 1 Day 5 • Note the sequence of the Antisense primer (see below) spans the second intron of the ACTB mRNA. Hence these primers should only produce a PCR product from 'full length' cDNA and will not be able to amplify genomic DNA. • A positive result will produce a 153-bp PCR product and indicate that the cDNA is suitable for PCR amplification Use PCR to amplify of coding sequence of gene of interest from cDNA • In this procedure you will you use PCR to amplify specific cDNAs created in the RT-PCR reaction Purify PCR products from gene of interest PCR reaction • You will use the Sigma spin column purification kit to purify the PCR products from the PCR reaction mixture Perform restriction digestion on purified gene of interest PCR products • You will use restriction enzymes to digest the PCR products creating a DNA sequence with ‘overhangs' that are compatible with the restriction digested plasmid ‘overhangs' Gel analyse restriction digested PCR products, and estimate ‘molar ratios’ for cloning • You will use an agarose gel to separate plasmid and PCR products aliquots • You will then estimate the concentration of the PCR products by comparing the intensities of bands visualised in the gel, using the ImageLab software NOTE: At some stage whilst the PCR is running, your group should prepare a 150 mL - 1% agarose/0.5X TAE gel PCR amplification of transcripts from cDNA The Pink PCR mix is Bioline MyTaq HS red PCR mix: catalogue number BIO-25047 PCR amplification of β-actin transcripts from cDNA 1. Add 12.5 μL of your cDNA into a 200 μL thin-walled PCR tube 2. Add 12.5μL of the pink solution in the 1.5mL tubes labelled A. This contains a x2 PCR master mix of MyTaq HS red PCR and 10 picomoles each of the Actin sense (forward) and antisense (reverse) primers PCR amplification of gene of interest transcripts from cDNA 1. Add 12.5 μL of your cDNA into a 200 μL thin-walled PCR tube 2. Add 12.5 μL of the pink solution in the 1.5mL tube labelled B. This contains a x2 PCR master mix of MyTaq HS red PCR and 10 picomoles each of a forward and reverse primer to amplify our the cDNA of our transcripts of interest Set a PCR thermal cycler to the conditions indicated below and ‘run’ your PCR. The reaction will take approximately 60 minutes to complete. Thermal cycler settings: Actin primer pairs (Tube A) Initial denaturation - 1 cycle: 95°C - 60s: Amplification - 35 cycles of {95°C - 15s, 55°C - 15s, 72°C - 10s} Final hold - 1 cycle - 10°C - hold Transcripts of interest primer pair (Tube B) Initial denaturation - 1 cycle: 95°C - 60s: Amplification - 35 cycles of {95°C - 15s, 59.2°C - 15s, 72°C - 15s} Final hold - 1 cycle - 10°C - hold After the PCR reaction Remove a 5 μL aliquot from the gene of interest PCR reaction into a clean 1.5 mL tube. Label the tubes with your student letter. Purification of PCR products from a PCR reaction The GenElute™ PCR Clean-up Kit is designed for rapid purification of single-stranded or double-stranded PCR amplification products (100 bp to 10 kb) from other components in the reaction, such as excess primers, nucleotides, DNA polymerase, oil and salts. DNA purification is achieved in a few easy steps. Purified DNA can be used in enzymatic reactions, conventional or automated sequencing, cloning and microarray analysis Video protocol: http://www.sigmaaldrich.com/video/biology/na1020.html 1. Add 5 volumes (100μL) of Binding Buffer to the PCR reaction, mix well and leave to stand for 1 minute 2. Take a GenElute PCR filter column and place it into a 2 mL collection tube 3. Pipette 500 μL of Column preparation solution into the top of the column 4. Centrifuge at 13,000rpm (12,000g) for 1 min 5. Discard the eluate from the collection tube, keep the GenElute column 6. Pipette the Binding Buffer/PCR mixture into the centre of GenElute column 7. Centrifuge at 13,000rpm for 1 min 8. Discard the flow-through from the collection tube into your waste bottle, keep the GenElute column 9. Pipette 500 μL of Wash solution into the centre of the GenElute column 10. Centrifuge at 13,000rpm for 1 min 11. Discard the flow-through from the collection tube into your waste bottle, keep the GenElute column 12. Centrifuge at 13,000rpm for 2 mins 13. Discard the flow-through from the collection tube into your waste bottle and the collection tube into a solid waste collection bag, keep the GenElute column 14. Place the GenElute column into the top of a clean labeled 2 mL collection tube. 15. Pipette 50 μL of Elution Solution into the centre of the GenElute column 16. Centrifuge at 13,000rpm for 1 min 17. Keep the collection tube (the eluate should contain the purified DNA). 18. Discard the GenElute column into a solid waste collection bag. 19. Remove 5 μL aliquot of the purified PCR product into a clean 1.5 mL tube. Label the tube Purified PCR Restriction enzyme digest of purified PCR products Setting up the restriction digestion reaction 1. Dispense 5μL of reaction buffer number into the tube containing the purified gene of interest PCR products (NOTE you SHOULD have 45 μL in the purification tube. CHECK THIS BEFORE YOU ADD THE BUFFER) 2. Bring the tube to a demonstrator and add 10 enzyme units of restriction enzyme to your tube (PstI and SgfI - buffer C) 3. Place the reaction in the 37°C heating block for at least 1hr. 4. Cool the tube to room temperature and proceed to gel analysis Agarose electrophoresis analysis of PCR and PCR/restriction digest products Preparing the samples for agarose gel analysis: 1. Remove 16μL of the restriction digest reaction into a clean 1.5mL tube and add 4μL x5 DNA loading dye 2. Mix with a fresh tip by pipetting the liquid up and down 2 - 3 times 3. Collect any drops of sample that may have adhered to the tube sides or lid to the bottom of the tube by 'flash-spinning' in a centrifuge 4. Remove 4μL of your ‘gel purified’ plasmid into a clean 1.5mL tube and add 1μL x5 DNA loading dye (from Week 1 - Day 3) Load the gel in the following order Lane 1: 5μL of a DNA ladder Lane 2: 5μL of Tube A PCR reaction (Actin) Lane 3: 5μL of Tube B PCR reaction, before purification Lane 4: 20μL of the Tube B PCR after purification, restriction digestion and addition of DNA loading dye Lane 5: 5μL of gel purified digested plasmid (from Week 1 - Day 3) Run the gel at 100 volts until the products are sufficiently separated (approximately 30-45 minutes should be sufficient) Week 2 Day 2: Ligation and transformation of E.coli with ligated plasmids The procedures you perform on this day one final objective To ligate restriction digested PCR fragments into plasmid vectors and then transform these ‘ligated' plasmids into chemically competent E.coli cells You will perform the following procedures. Ligate restriction enzyme digested PCR products into prepared plasmid • In this procedure you will ligate the digested PCR fragments into the linearised gel purified plasmid • The ligation reaction should create 'recombinant’ plasmids containing PCR fragments in the linearised plasmid restriction sites Transform ligated plasmid::’transcripts of interest' plasmids into chemically competentE.coli cells • You will perform 2 transformations; one a ligation reaction and one control reaction • In the transformation procedure you will add a small aliquot of your ligation reaction and control reaction to chemically competent E.coli cells. The transformation process will facilitate the E.coli cells to ‘take up’ plasmids. Single E.coli cells that are successfully transformed will be antibiotic resistant and therefore able to grow into colonies of cells on the LB antibiotic plates • The control reaction transformation will test transformation efficiency of the cells i.e. how well the E.coli cells 'take up' a known amount of circular plasmid. The results of this will also show how well you performed the transformation experiment • The transformation using the ligation reaction will facilitate the E.coli cells to ‘take up’ any ligated-plasmids present/formed during the ligation reaction. Ligate PCR products into prepared plasmid You have been supplied with one 1.5 mL microfuge tube labelled L (for ligation) and one 1.5mL microfuge tube C (for control). Assemble ligation and control reactions in the order below. Tube C Control reaction • This tube contains 1μL (≈25ng) of undigested plasmid • Add 15μL ultra pure water to the tube • Add 4μL of 4 x QS Buffer to the tube and mix thoroughly by gentle pipetting up and down 3-4 times. • Collect all liquid to the bottom of the tube, by flash spinning Tube L Ligation reaction • Add 1μL of gel purified plasmid (≈25g of DNA) • Add 14μL of the purified pooled MFBK PstI and SgfI digested PCR reactions to the tube • Add 4μL of 4x QS Buffer and mix thoroughly by gentle pipetting up and down 3-4 times • Collect all liquid to the bottom of the tube, by either flash spinning and/or gentle tapping on a hard surface • When you have created the above reactions bring both your tubes to the demonstrator who has the QS Ligase enzyme on ice. Add 1μL of Bioline QS Ligase to the ligation reaction tube. This will 'start' your ligation reaction. • Incubate both the ligation and control tubes at room temperature for no longer than 5 minutes. • After incubation, place both reactions on ice and immediately proceed to carry out the transformation procedure. Transform ligation reaction plasmids into chemically competent E.coli cells Video introduction to Transformation using commercially available competent cells • Thaw 50μL of Chemically Competent cells on ice for 10 minutes. • Pipette 25μL of the thawed cells into a clean sterile 1.5mL microfuge tube and label the tube control • Pipette 25μL of the thawed cells into a clean sterile 1.5mL microfuge tube and label the tube ligation • Pipette 5μL of control mixture into the control cells tube and carefully flick/tap the tube 5 times to mix. Do not vortex. • Pipette 5μL of ligation mixture into the cells and carefully flick the tube 5 times to mix. Do not vortex. • Place both tubes on ice for 30 minutes. Do not mix. • Heat shock the cells in both tubes in a water bath set to exactly 42°C for exactly 30 seconds. Do not mix. • Place both tubes on ice for 5 minutes. Do not mix. • Pipette 270μL of room temperature SOC media into both tubes. • Incubate both tubes at 37°C with shaking at 250 rpm for 60 minutes. • After incubation mix the cells thoroughly by flicking the tube • Onto the plate labeled control 5, pipette 5μL of the control ligation cells + 95μL of SOC media • Onto the plate labeled control 100, pipette 100μL of the control ligation • Use a sterile spreader to spread the cells evenly over both agar plates. 'Spread' the control 5 plate first • Onto the plate labelled L20, pipette 20μL of cells + 80μL SOC media • Onto the plate labelled L40 pipette 40μL of cells + 60μL SOC media • Onto the plate labelled L80 pipette 80μL of cells + 20μL SOC media • Onto the plate labelled L160 pipette 160μL of cells • Use a single sterile spreader to spread the plates in the following order: first the L20 plate, second the L40 plate, thirdly the L80, and lastly the L160 plate. Take care to spread the cell solutions evenly and gently onto the agar plate surfaces. Video: Example of how to 'spread your plates • Seal (in plastic bags) the plates and incubate at 37°C, at least overnight You will analyse potentially recombinant 'positive’ E.coli colonies by colony PCR to detect potential recombinant plasmids Week 2 Day 3: Colony PCR to detect potential recombinant plasmid positive E.coli colonies The procedures you perform on this day one final objective. To detect E.coli cells that contain recombinant plasmids with inserts You will perform the following procedures. Perform a set of colony PCR reactions • In this procedure you should choose 4 white E.coli colonies from the ligation plates and perform a colony PCR procedure on each colony to detect potential cloned inserts The PCR primers flank the multicloning restriction site on the plasmid will amplify any DNA which lies between them (see images at bottom of page). Use agarose gel electrophoresis to analyse colony PCR reaction Products • You will use a 1% agarose gel to separate PCR products Colony PCR is a useful way to 'quickly' to screen multiple bacterial colonies to see if any contain recombinant plasmids with the approximately the expected DNA insert 'size'. The principle relies upon the ability of the PCR buffer and heating in the thermal cycler to burst open bacteria, releasing their contents including any plasmid DNA molecules they may contain. The procedure will use a pair of plasmid specific primers to amplify any DNA fragments that have been ‘inserted’ into the multi-cloning site of a plasmid during the ligation reaction carried out on Week 2 Day 2. The primers the amplify the ‘insert’ anneal to a region 5′ and 3′ to the multi-cloning site of the plasmid. Creating a PCR master mix for colony PCR and selecting E.coli colonies • Label two rows of 200μL PCR tubes with your student letter and a number e.g. A1, A1, A3, A4 • Place them opposite each other in the orange holder • Aseptically dispense 100μL of LB antibiotic broth into each of the tubes of the second row • Into the first tube of the first row, add 10 μL of the plasmid specific primers*, 40μL of ultra pure ddH2O and 50μL of x2 Bioline MyTaq HS red PCR mix into the tube and mix by gently pipetting up and down 3-4 times. • Next aspirate 25μL of the tube 1 'master mix' into the 3 remaining tubes of row 1 • Next, fit either a p10 or p20 with a fine point sterile tip and gently touch the surface** of an E.coli colony you wish to colony PCR check. Choose ONLY single white colonies that are growing in a area of the plate ‘free’ of any blue colonies • Next, 'dip' the tip into the first tube of row 1 master mix and then eject the tip into the first tube of row 2 (containing the LB ampicillin broth). Repeat this process until you have selected and transferred 3 more colonies into PCR mix and LB ampicillin broth tubes • Place your LB ampicillin broth strip into a 36°C incubator for at least 1 hr • Place your PCR master mix 'strip' into the thermal cycler set to the conditions indicated below and ‘run’ your PCR. The reaction will take approximately 65 minutes to complete. Initial denaturation - 1 cycle: 95°C - 3 mins Amplification - 40 cycles of {95°C - 15s, 55°C - 15s, 72°C - 20s} Final hold - 1 cycle - 10°C - hold **As you touch the surface of the colony a number of bacteria will 'stick' to the tip. As you 'dip' the tip into the first PCR tube and then 'eject' it into the second some of the will then be transferred into each tube. This step is part of 'the art' of colony PCR. You don't want too many bacteria in the PCR master mix or the PCR may be inhibited. You also don't want too few or the PCR may not be able to amplify the DNA. You also need 'enough' bacteria in the LB to grow. This is usually not a problem though. Agarose gel analysis of results At a convenient point during the day, when the PCR is running, each group should prepare a 1% agarose gel Loading the gel: The MyTaq HS PCR master mix already contains a loading dye, that is why it is 'pink’. So you do not need to add a DNA loading dye Load your samples into the gel in the following order Lane 1: 5μL of DNA ladder Lane 2: 5μL tube 1 colony PCR Lane 3: 5μL tube 2 colony PCR Lane 4: 5μL tube 3 colony PCR Lane 5: 5μL tube 4 colony PCR For each insert positive colony PCR, transfer all 100μL of the inoculated LB ampicillin broth from a strip tube into a 15mL Falcon tube containing 5 mL of LB antibiotic broth. Incubate the Falcon at 36°C incubator over night in preparation for plasmid extraction Example colony PCR results. In this case if a colony PCR gives a results similar to that shown below, this would be a good indication the the colony PCR loaded in lane 1 came from a colony that potentially has a full length insert cloned. Introduction and Class Results Week 2 Day 4: PCR fragment restriction digest analysis The procedures you perform on this day have one final objective To confirm the ability of the PstI and SgfI restriction enzymes, in buffer C, to digest a liner PCR fragment produced from the RC214690 TROVE2 (Myc-DDK-tagged)-Human TROVE transcript variant 1 plasmid You will perform the following procedures. Purification of PCR products from a PCR reaction • You will use a commercial spin column purification kit to purify PCR fragments created from the colony PCR • You will then determine the concentration of your purified DNA using the Qubit Perform a restriction enzyme digest on purified PCR fragment • You will use the PstI and SgfI restriction enzymes to digest the purified PCR to determine the digest pattern Use an agarose gel to analyse restriction digested plasmid to confirms • You will use a 0.8% agarose gel to separate restriction digest fragments • You will use ImageLab software to determine fragment sizes of digested PCR fragments Purification of PCR products from a PCR reaction The GenElute™ PCR Clean-up Kit is designed for rapid purification of single-stranded or double-stranded PCR amplification products (100 bp to 10 kb) from other components in the reaction, such as excess primers, nucleotides, DNA polymerase, oil and salts. DNA purification is achieved in a few easy steps. Purified DNA can be used in enzymatic reactions, conventional or automated sequencing, cloning and microarray analysis Video protocol: http://www.sigmaaldrich.com/video/biology/na1020.html 1. Add 5 volumes (100μL) of Binding Buffer to 20μL of one of the colony PCR reactions, Mix well and leave to stand for at least 1 minute 2. Take a GenElute PCR filter column and place it into a 2 mL collection tube 3. Pipette 500μL of Column preparation solution into the top of the column 4. Centrifuge at 13,000rpm (12,000g) for 1 min 5. Discard the eluate from the collection tube, keep the GenElute column 6. Pipette the Binding Buffer/PCR mixture into the centre of GenElute column 7. Centrifuge at 13,000rpm for 1 min 8. Discard the flow-through from the collection tube into your waste bottle, keep the GenElute column 9. Pipette 500μL of Wash solution into the centre of the GenElute column 10. Centrifuge at 13,000rpm for 1 min 11. Discard the flow-through from the collection tube into your waste bottle, keep the GenElute column 12. Centrifuge at 13,000rpm for 2 mins 13. Discard the flow-through from the collection tube into your waste bottle and the collection tube into a solid waste collection bag, keep the GenElute column 14. Place the GenElute column into the top of a clean labeled 2 mL collection tube. 15. Pipette 50μL of Elution Solution into the centre of the GenElute column 16. Centrifuge at 13,000rpm for 1 min 17. Keep the collection tube (the eluate should contain the purified DNA). 18. Discard the GenElute column into a solid waste collection bag. Determining the concentration of purified PCR DNA Using the Invitrogen Qubit flourometer. Use these video link guides 1 and 2 for information on how to carry out the protocol Protocol • An aliquot of working reagent, sufficient to carry out 16 estimations will have been prepared • For your sample analysis, dispense 5μLs of purified PCR DNA into tube S and 195 μL of Quant-IT working solution • Vortex all the tubes for 2-3 seconds and then incubate for 2-5 minutes at room temperature. • Use the Qubit to determine the DNA concentration in your sample. The standards will have been made for you • Record your values in your lab book and the Class Results Excel file. Perform a restriction enzyme digest on purified PCR fragment In this procedure you will digest 100ng of your purified PCR fragment, using the PstI and SgfI restriction enzymes. You will prepare one digestion reaction and one control reaction. NOTE: At some stage during an incubation, your group should prepare a 150 mL - 0.8% agarose TAE gel with GelRed dye Setting up the control reaction • Label a clean 1.5 mL microfuge tube C (for control). • Add 100ng of your purified PCR reaction • Dispense XμL of ddH2O into the tube to bring the volume to 18μL • Dispense 2μL of enzyme buffer C into the tube • Mix by gentle pipetting, flash spin to collect all the reagents to the bottom of the tube Setting up the digestion reaction • Label a clean 1.5 mL microfuge tube D (for digest). Then add the number of each of your purified plasmids • Add 300ng of your purified PCR reaction • Dispense XμL of ddH2O into the tube to bring the volume to 17μL • Dispense 2μL of enzyme buffer C into the tube • Mix by gentle pipetting, flash spin to collect all the reagents to the bottom of the tube • Bring the tube to a demonstrator and add 0.5μL each of restriction enzymes mix to your tube. • Incubate all tubes in the 37°C heating block for at least 60 minutes up to 2hr maximum. Record how long you digested your DNA sample. Use an agarose gel to analyse restriction plasmid to generate a 'plasmid map’ for every plasmid Preparing the samples for agarose gel separation: • Add 5μL x5 DNA loading dye into each of the reaction tubes (control and digest) • Mix with a fresh tip by pipetting the liquid up and down 2 - 3 times • Collect any drops of sample that may have adhered to the tube sides or lid to the bottom of the tube by 'flash-spinning'. Load samples into the gel in the following order Lane 1: 5 μL of a DNA ladder. Lane 2: all the control reaction Lane 3: all of the digest reaction Run the gel at 150 volts until the products are sufficiently separated (approximately 45 minutes should be sufficient). Image the gel using the GelDocEZ system and analyse the results using the ImageLab system