Team:UCL/Science/Proto
From 2014.igem.org
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<div id="view8"><div class="textTitle"><h4>Standard PCR Protocol</h4></div><br> | <div id="view8"><div class="textTitle"><h4>Standard PCR Protocol</h4></div><br> | ||
<p>Although our PCR protocol evolved throughout the project this protocol represents the core procedure we used throughout the project.</p><br> | <p>Although our PCR protocol evolved throughout the project this protocol represents the core procedure we used throughout the project.</p><br> | ||
<p><b>1. Make stock solution primer mix</b><br> | <p><b>1. Make stock solution primer mix</b><br> | ||
- | Make 1/10 dilution of forward and reverse primer mix with TE buffer solution or Milli-Q water (e.g 10uL forward primer, 10uL reverse primer and 180uL TE buffer). Label with primer names, concentration, date and name of user.<br></p> | + | Make 1/10 dilution of forward and reverse primer mix with TE buffer solution or Milli-Q water (e.g 10uL forward primer, 10uL reverse primer and 180uL TE buffer). Label with primer names, concentration, date and name of user.<br></p><br> |
<p><b>2. Plan the PCR</b><br> | <p><b>2. Plan the PCR</b><br> | ||
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Use this calculation to calculate the volumes of forward and reverse primers to be used in the PCR. | Use this calculation to calculate the volumes of forward and reverse primers to be used in the PCR. | ||
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+ | <p><b>3. PCR Program:</b></br></br> | ||
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Revision as of 00:57, 18 October 2014
![Protocols](https://static.igem.org/mediawiki/2014/4/41/OProtocols_Bannero.jpg)
- Creating competent cells
- Digestion
- Ligation
- Making LB Agar Plates
- Transformation
- Agarose Gel Electrophoresis
- Colony Boil
- Standard PCR Protocol
Creating competent cells
LB Media, 50ml Falcon Tubes, Ice, Chilled centrifuge, Calcium Chloride (CaCl2), Eppendorf tubes (300ul/tube)
Procedure
1. Inoculate a single colony into 5ml Lb in 50ml falcon tube. Grown O/N @ 37oC
2. Use 1ml to inoculate 100ml of LB in 250ml bottle the next morning.
Shake @ 37oC for 1.5-3 hours.
Or
1. Inoculate a single colony into 25ml LB in a 250ml bottle in the morning
2. Shake @ 37oC for 4-6 hours.
Then…
3. Put the cells on ice for 10mins (keep cold from now on).
4. Collect the cells by centrifugation in the big centrifuge for 3 minutes @ 6Krpm.
5. Decant supernatant and gently resuspend on 10ml cold 0.1M CaCl (cells sensitive to mechanical disruption).
6. Incubate on ice x 20 minutes
7. Centrifuge as in 2.
8. Discard supernatant and gently resuspend on 5ml cold 0.1M CaCl/15%Glycerol.
9. Dispense in microtubes (300ųl/tube). Freeze at -80oC.
Note: Home-made competent cells were used to transform registry BioBricks
Digestion
MilliQ water, NEBuffer 2.1, BSA, Pipettes and autoclaved tips Enzymes: EcoRI-HF, SpeI, XbaI and PstI,
Notes
eg. if concentration BBa_R0010 is available at a concentration of 330ng/ul
we require 500/330 = 1.5ul of the DNA solution for a digestion
For Upstream parts, such as promoters digest with EcoRI-HF and SpeI
Digest downstream parts with XbaI and PstI
Digest plasmid backbones with EcoRI-HF and PstI
Procedure
1. Calculate amount of DNA solution that will be needed for 500ng
2. Following this, calculate the amount of water needed to make up to 50ul if both enzymes and BSA are 1ul each and Buffer is 5ul.
3. Add the components to a microcentrifuge tube in the order: Water > Buffer > BSA > DNA > Enzymes
4. Leave to incubate at 37ºC for 1-2 hours
5. Remove and deactivate the enzymes by incubating at 80ºC for 20 minutes
Ligation
MilliQ water, Ice, Pipettes and autoclaved tips, NEB T4 DNA Ligase Buffer, T4 DNA Ligase
Notes
Procedure
1. Calculate water needed to total volume of 20ul where enzyme added will be 1 ul and all other components will be 2ul
- Usually this will include buffer, the upstream part digestion, downstream part digestion and backbone digestion.
2. Do no-insert and no-vector control ligations for each backbone and insert
3. Add components in the order: Water > Buffer > DNA upstream part > DNA downstream part > Backbone DNA > Ligase
4. Leave for 10 minutes at room temperature approx. 25ºC
5. Deactivate at 80ºC for 20 minutes
6. Transform products of ligation into competent cells
Making LB Agar Plates
Materials
LB Agar powder, plates, antibiotics, sterile jar, sterile flame or biosafety cabinet.
Procedure
1. Dissolve LB agar in sterile/autoclaved jar of sterile water 40g/L.
2. Swirl until completely dissolved
3. Autoclave LB agar
The following steps should be completed under a sterile flame or biosafety cabinet. Ideally after autoclaving, before LB Agar solidifies:
4. Add antibiotics from their stock solutions in 1:1000 ratio with LB agar, can be added either directly to each individual plate or to the entire solution.
5. Pour agar into plates, roughly half way (30-50mL per plate). Leave plates with lids half on (prevent condensation).
6. Allow plates to solidify under flame
7. Cover and leave on bench or in fridge
Plasmid DNA Transformation
Materials
Competent Cells, Plasmid DNA, Antibiotic Plates
Procedure
1. Thaw competent cells on ice
2. 50uL cells enough for 1 transformation
3. Add 1ug of DNA to 50uL competent cells
If biobrick from distribution, resuspend DNA well in 10uL ddH20
4. Add 2-5uL DNA i.e. ligation product or BioBrick re-suspension to 50uL competent cells
5. Flick by hand or pipette up and down gently
6. Place cells on ice for 30 minutes
7. Place cells in water bath or heat block at 42oC for 30 seconds
8. Place cells on ice for 2 minutes
9. Add 950ul of SOC or LB media and place in a shaking incubator for a maximum of 2 hours (37oC/250rpm)
10. Label two petri dishes with LB agar and the appropriate antibiotics(s) with the date, plasmid backbone and ligated genes/parts and antibiotic added.
You may also choose to write down the volume of transformed product plated
11. Spin down the incubated cells for 2mins at 4000rpm to obtain a cell pellet.
12. Pipette 100ul of the supernatant or fresh LB media before discarding the rest of the supernatant and re-suspending the pellet in the 100ul of media
13. Incubate the plates at 37ºC for 12-14 hours, always place plates lid-down.
If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria
You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.
Count the colonies on the 20 μl control plate and calculate your competent cell efficiency.
Agarose Gel Electrophoresis
Prepare Agarose Gel (1%) for Electrphoresis
Materials:
Agarose powder, spatula, weighing boat, microwave, Ethidium Bromide, Gel dock,
1. Add ingredients to flask and microwave for about 120 seconds. Shake. Repeat until mixture fully dissolved.
2.Leave to cool for 20-30 seconds.
3.Add 20ul of Ethidium Bromide.
Place comb in gel frame. When flask is cool enough to handle, pour the solution into the gel frame and leave to set (10-20 minutes).
Preparing for Loading:
1. Prepare Ladder
- a. Add 1ul of DNA ladder to labelled Eppendorf.
- b. Add 1ul of DNA loading buffer (dye) to Eppendorfs containing DNA and DNA ladder (Unless ladder is already combined with dye).
- c. Add 1ul of milliQwater
- d. Spin down.
2. Prepare samples:
- a. Add 10ul of digest (250ng of DNA) to 3ul of loading buffer.
Prepare Ladder
- 1. Place gel frame in electrophoresis box with black (cathode) part away from you and red (anode) part closest to you.
- 2. Carefully remove comb from gel.
- 3. Fill the top and bottom wells with 1x TAE. Pour over top of gel, continue pouring until gel is covered.
- 4. Load the wells starting with the DNA ladder in lane 1. Be sure to note which lane each sample is loaded into.
- 5. Run gel for approximately 60 minutes at constant voltage of 120V. Check for bubbles at the cathode which shows it has started.
- 1. Remove the frame from the electrophoresis box and pour off any liquid. Wipe the bottom of the frame with tissue.
- 2. Place in GelDoc 2000, press Epi white to position gel, press Epi UV to visualise.
- 3. On the computer open program Quantity One. Select scanner, GelDoc XR, select manual acquire, adjust value until gel is visible.
- 4. Print or save to USB.
- TAE from the gel run can be re-used, pour back into bottle.
- Ensure all ethidium bromide rubbish is disposed of in ethidium bromide bin.
Colony Boil
Colony boil can be used to isolate genes present in an organism. We used a colony boil to isolate AzoR from e.coli using primers originally designed for PCR.
1. Using a sterile pipette tip carefully lift a single colony from any most recently transformed E.coli plate.
Repeat for more colonies from different strains if available.
2. Point the tip into 250ul of distilled/MilliQ water in an eppendorf, MUST BE screw-cap, and shake a bit. Withdraw and discard tip.
3.Place the tube in a 100͒C water bath for 5 minutes.
4. Remove and dry the outside of the tube. Shake the contents of the tube to the bottom. Use 2ul of the boiled cell solution for PCR with genomic DNA primers for any genes of interest that are present in the genome such as IspB
Standard PCR Protocol
Although our PCR protocol evolved throughout the project this protocol represents the core procedure we used throughout the project.
1. Make stock solution primer mix
Make 1/10 dilution of forward and reverse primer mix with TE buffer solution or Milli-Q water (e.g 10uL forward primer, 10uL reverse primer and 180uL TE buffer). Label with primer names, concentration, date and name of user.
2. Plan the PCR
V1 x C1=V2 x C2 where V= volume in uL, C=concentration in uM for each of the primers.
Use this calculation to calculate the volumes of forward and reverse primers to be used in the PCR.
3. PCR Program:
Stages | NEB Phusion DNA Polymerase | Roche Taq DNA Polymerase | PROMEGA Pfu DNA Polymerase |
---|---|---|---|
Hot start (oC) / time | 98oC / 30s | 94oC / 2min | 95oC / 1-2min |
Cycles(#) | 25-35 | 25-30 | 25-35 |
Denaturation (oC) / time | 98oc / 5-10s | 94oC / 15-30s | 95oC / 30s-1min |
Annealing (oC) / time | 45-72oC / 10-30s | 55 to 65oC / 30-60s | 42-65oC / 30s |
Elongation (oC) / time | 72oC / 15-30s per kb | 72 OR 68oC / 45s - 2min | 72-74oC / 2-4min |
Final Extension (oC) / time | 72oC / 5-10min | 72 OR 68oC / 7min | 72-74oC / 5min |
Hold temp (oC) / time | 4 to 10oC / infidenlity | 4oC / infidelity | 4oC |