"
Contents |
Colony PCR of pcDNA3.1(+)-LovTAP (Part 1)
Plan : Use T7 + BGH and T7 + LovTAP check primer sets to check the presence of LovTAP in our colonies and verify that the backbone is pcDNA3.1(+) and not LovTAP's former backbone pMA (determined by the presence or not of a polyA signal BGH, which is absent in pMA).
Since the Tm for T7 was nowhere to be found, a gradient PCR was made to figure it out. [insert picture of gel here and maybe protocol]
Colony preparation: The four plates were labeled
- A (pcDNA3.1-LovTAP unpurified 1:2)
- B (pcDNA3.1-LovTAP unpurified 1:3)
- C (pcDNA3.1-LovTAP purified 1:3)
- Control (pcDNA3.1 only)
Of each one we decided to pick 8 colonies, except for the control where we picked only one.
- 5-10ul of Lyse and Go was added into all the destination PCR tubes
- 3ml of LB + 80ul of 100mg/ml Amp in the culture tubes
- Colonies were picked, dipped into the culture tube and then into the PCR tube.
- Culture tubes were incubated at 37°C and PCR tubes stored in Matt's fridge until next morning.
Protocol: None
Forgot to insert protocol.
PCR product digestion/purification
The following PCRs worked at Matt's lab: SEAP, TNFR and eGFP (also used as a control at a Tm of 55°C). Therefore these products were ready to be digested in order to be ligated into pGL4.30 to achieve the cloning of the three Fussenegger experiment readouts. Digestion:
- TNFR and eGFP with HindIII and FseI
- SEAP with HindIII and MfeI
Protocol: Quick and Dirty Digestion
Since Nanodrop concentration results for PCR and samples extracted from gels have proved unreliable, we have arrived at a sort of a default protocol for DNA of an uncertain concentration.
For a ligation or when large quantities of DNA are needed:
- DNA 15 microliters
- Restriction enzyme 1 @ 100x 1 microliters
- Restriction enzyme 2 @ 100x 1 microliters
- BSA (if needed) @ 10 x 5 microliters
- Demineralized water 28 microliters
For checking the presence of a restriction site on a gel:
- DNA 5 microliters
- Restriction enzyme 1 @ 100x 1 microliters
- Restriction enzyme 2 @ 100x 1 microliters
- BSA (if needed) @ 10x 5 microliters
- Demineralized water 38 microliters
The total volume should be 50 microliters
Incubation should ideally be at 37°C. When in doubt on the quantity of DNA used, favor a longer (20 to 30 minutes longer) incubation time than what is indicated on the NEB double digest guide. Avoid enzyme pairs with different incubation temperatures. This doubles the incubation time.
After the incubation period, the enzymes need to be heat-inactivated. Heat inactivation temperatures are usually on the order of 80 C. 20 minutes usually does the job.
The samples can then be frozen if needed.
Two separate tubes were digested for each readout, one of which was purified (PCR clean up kit protocol) afterwards. That way we will be able to compare the results between ligation with or without previous PCR clean up and see if it's a necessary step or not.
- Comments
Insert comments about what happened.
LovTAP readout gel extraction
In order to clone the LovTAP readout into a mammalian expression vector (either pcDNA3.1(-) or pCEP4) we can simply cut it out with KpnI and BamHI. Alternatively, KpnI and XhoI also work but only for pcDNA3.1(-) insertion. So two different digestions were made on the Maxiprep of the LovTAP readout (RO):
- KpnI + BamHI
- KpnI + XhoI
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).
The two products were run on a 1% agarose gel and the lower band was then cut out and purified with the gel extraction kit following the protocol in the manual.
- Comments
Insert comments about what happened.
Agar plate preparation
We used all the Ampicillin agar plates for previous transformation, therefore we had to prepare new ones.
Protocol: None
Forgot to insert protocol.
Write protocol for Agar plate preparation.
- Comments
Insert comments about what happened.
