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PCR optimisation
Protocol: PCR
PCR is a reaction that makes it possible (and relatively easy) to amplify
a certain region of DNA. The first step is the selection of that region
(and the design of the relevant primers). Primer design can be done by hand, or by
using our Primer Design Helper. Once
done, order the primers (in our case, we ordered from them IDT).
When you've received the primers, prepare them and make sure you've got your PCR kit (we used the "Phusion® High-Fidelity DNA Polymerase"). Start preparing your master mix, the composition for one tube is:
1X Mastermix 20μl reaction, add in this order
| Reagent | Volume [μl] |
|---|---|
| Water | Complete to total volume of 20μl |
| HF-Buffer (5x) | 4 |
| DMSO (optional) | 0.6 |
| dNTPs | 0.4 |
| Forward primer (50μM) | 0.2 |
| Reverse primer (50μM) | 0.2 |
| Template (10ng/μl) | 0.5 |
| Phusion HF polymerase | 0.2 |
Prepare one or two extra tubes-worth of reagent (you'll use some liquid on the walls of your tips).
Once you've finished, you should run the resulting products on a gel to check if everything went as planned.
Tips
- Thaw the HF-Buffer, DMSO and dNTPs before making the mastermix.
- Avoid taking the Phusion-HF polymerase out of the freezer (only take it out briefly when you need to add it).
- If the reactions have different primers and/or template, add the polymerase right after the dNTPs, split the mastermix and add the rest.
- Don't forget positive and negative controls
- Primers should have similar Tms (less than 5°C).
- Primer Tm calculation is a less exact science than it should be (just test several tools and compare their results). If you're not sure what the correct Tm is, consider using a gradient PCR.
- Avoid primers with strong secondary structures.
- PCR can introduce mutations. Don't forget to sequence your final product (this could be your final plasmid): you really don't want to lose a few weeks because of a "corrupt" plasmid.
In order to figure out why our PCRs give very random successful results, we thought about trying to compare between HF-Buffer protocol and GC-Buffer protocol and adding DMSO to both. We also added the four apparently successful pMP-LovTAP ligations to the templates, in order to check (in case one of the PCRs works) if we really have a LovTAP gene in the plasmid.
PCR'd genes: LovTAP, GFP, SEAP, TNFR.
Templates
- pMA-LovTAP
- pMP-LovTAP (L2)
- pMP-LovTAP (L3)
- pMP-LovTAP (L5)
- pMP-LovTAP (L9)
- pMP-PB IRES GFP
- pMP-PB SEAP-FLAG with pGL4.30 insertion primers
- pMP-PB SEAP-FLAG with Biobrick primers
- pMP-PB-TNFRFc with pGL4.30 insertion primers
- pMP-PB-TNFRFc with Biobrick primers
pMP-LovTAP digestion
Protocol: Restriction site digestion
- Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
- NEBcutter for finding cutting enzymes.
- Double Digest Finder for the parameters.
- Calculate the amounts required of:
- DNA
- Buffer (usually from 10x to 1x)
- BSA, if needed (usually from 100x to 1x)
- Enzymes (depends on the amount of DNA)
- Water
- Get the recommended buffer (and BSA if needed) from the freezer and let defreeze.
- Mix all the ingredients, except DNA, in a tube.
- Note: Enzymes should stay no longer than a couple of minutes out of the freezer. Don't touch the bottom of the tubes! Don't vortex!
- Distribute the mix in as many tubes as DNA samples and add the DNA.
- Keep in the Thermomixer at the recommended temperature.
Sowmya's recommended amounts (50 µl total solution):
- 5 µl of 10x buffer
- 0.5 µl of 100x BSA
- 1 µl of each enzyme
- 5 µl of DNA
- 37.5 (up to 50 µl) of water.
Protocol based on what was done on July the 4th.
We also performed another digestion on pMP-LovTAP to check the nature of the backbone this time.
- Master Mix for 4 digestions of pMP-LovTAP with NotI/SpeI (196 µl total)
- H20: 144 µl
- N2 buffer: 20 µl
- BSA 10x: 20 µl
- NotI: 4 µl
- SpeI: 4 µl
Split in 4 tubes, add 2 µl of the LovTAP-pMP miniprep (here, L2, L3 L5, L9). Incubate at 37°C for one hour, inactivate the enzyme, run on a gel along with the PCR products.
Gel electrophoresis of PCR products and of the pMP-LovTAP digestion
Protocol: Gel Electrophoresis
Agarose concentration depends on the size of the DNA to be run. We will mostly use 1%.
VOL is the desired volume of gel in ml:
CH Lab
- Add 0.01*VOL g of agarose to a clean glass bottle.
- Pour VOL/50 ml of 50xTAE in a graduated cylinder. Fill up to VOL ml with di water.
- Add the resulting VOL ml of 1xTAE to the glass bottle with agarose.
- Microwave, at 7, the bottle (loose cap!) until it boils.
- Carefully remove bottle (can be super heated!) and check for the total absence of particles. Microwave again if needed.
- Prepare a gel box, with comb, and fill it up with the agarose solution (maybe not the whole solution is needed).
- Add 0.05 µl per ml of gel in the box of Red Gel (it's in the iGEM drawer) and stirr until disolved.
- Wait until cold and solidified.
- Carefully remove comb.
- Place the box in the electrophoresis chamber.
- Fill up the electrophresis chamber with 1x TAE buffer.
- Add blue dye to the DNA samples (6x loading buffer, that is 10 µl in 50 µl of DNA solution).
- Inject 30 µl of ladder marker in the first well (that's 1 µg of DNA).
- Inject 60 µl of each DNA solution in the other wells.
- Set voltage to 70-90 V and run for 30-40 min, or until the dye reaches the last 25% of the gel length (DNA travels from - to +).
- Place the gel under the camera, cover, turn UV on and take photos!
Preparing the ladder:
- get 1kb ladder DNA from the freezer (500 µg/ml).
- for 30 charges, 30 µl per charge, we need 900 µl:
- 60 µl of 1kb ladder DNA
- 150 µl of dye (6x loading buffer)
- 690 µl of water
BM Lab
In this lab the gels are slightly different. The total volumes for the small, the medium and the large gel are respectively 60ml, 80ml and 90ml. As we use 0.5x TAE buffer instead of 1x, we can use higher voltages (170V seems to work fine). The gel should run 20-40 minutes, not more. As the gel is thinner, load less DNA (up to ~10ul).
- Gel image
%2Bdigestion_of_supposed_LovTAP-pMP.jpg)
We seem to have obtained LovTAP PCR products of the correct size based on every template. The backbone of this plasmid, however, seems weird. The HF buffer didn't work and the GC buffer did.
pMP-LovTAP transformation
Protocol: E.Coli Transformation
- Thaw the competent E.coli (DH5alpha) cells on ice (not in hands!)
- As soon as it is thawed, add 50µl of the cells to the DNA (~50-100 ng of pure plasmid, or some 2 µl usually)
- Let it rest on ice for 20-30 min. Meanwhile, put agar plate (with correct antibiotic) at 37°C for prewarming.
- Put the tube with DNA+E.coli at 42°C for 45 sec - 1 min (heat shock)
- Add 400 µl of LB broth and place at 37°C for 20-30 min (shaking)
- Spread the cells on the prewarmed plate (and let it dry)
- Incubate the plate upside-down at 37°C for ~14-15 hours (leaving it more than 16h decreases the plasmid quality)
Second attempt to transform our ligation product without E.Coli overgrowth.
