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Observation of pMP-LovTAP cultures
Growth in all tubes except negative control.
Miniprep
Protocol: Miniprep
The slim tubes can be centrifuged in the machine in front of the "Gel hood", at 4000 rpm for 10 min. The fatter ones, in the E. coli centrifuge by the fridge (the tip can be left inside, since it floats).
Pellets resuspended with RNase containing buffer (Resuspension Buffer R3, from Invitrogen, equivalent to Buffer P1 from Qiagen, in Sowmya's box in the fridge). Note: keep the buffer in ice if you are not bringing it back to the fridge for some minutes.
We then use the QIAGEN QIAprep Spin Miniprep Kit with their protocol (page 22) and a microcentrifuge.
Minipreps were made out of every LovTAP culture.
Digestion of pGL-TNFR and of pGL-SEAP
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.
- pGL4.30-TNFR with EcoNI
Since the gel from the day before was extremely unconvincing (we're not even sure of the sizes), we made a new digestion check. This time we picked a site that exists inside TNFR, but not in the backbone: EcoNI.
- Master Mix for 12 digestions of pGL4.30-TNFR with EcoNI (600 µl total)
- H20: 514 µl
- N4 buffer: 60 µl
- EcoNI: 2 µl
Then split into 12 tubes and add 2 µl of the TNFR-pGL ligation to every one of them. The digestion incubated for one hour. The enzyme was inactivated by 20 minutes at 65°C and a gel was prepared.
- pGL4.30-SEAP with NdeI
A "unique cut" enzyme (inside of SEAP) was also chosen to check if the pGL-SEAP ligation had worked.
- Master Mix for 10 digestions of pGL4.30-SEAP with NdeI (500 µl total)
- H20: 428 µl
- N4 buffer: 50 µl
- NdeI: 2 µl
Then split into 10 tubes and add 2 µl of the SEAP-pGL ligation to every one of them. The digestion incubated for one hour. The enzyme was inactivated by 20 minutes at 65°C and a gel was prepared.
Gel electrophoresis
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).
- pGL4.30-TNFR with EcoNI
Every digestion of pGL-TNFR ran on the gel next to its supercoiled (non-digested) version. The gel scheme was as follows:
- Top of the gel
- Lane 1: ladder
- Lane 2: TNFR1 coiled
- Lane 3: TNFR1 digested
- Lane 4: TNFR2 coiled
- Lane 5: TNFR2 digested
- Lane 6: TNFR3 coiled
- Lane 7: TNFR3 digested
- Lane 8: TNFR4 coiled
- Lane 9: TNFR4 digested
- Lane 10: TNFR5 coiled
- Lane 11: TNFR5 digested
- Lane 12: ladder
- Bottom of the gel
- Lane 1: ladder
- Lane 2: TNFR6 digested
- Lane 3: TNFR7 coiled
- Lane 4: TNFR7 digested
- Lane 5: TNFR8 coiled
- Lane 7: TNFR8 digested
- Lane 8: TNFR9 digested
- Lane 9: TNFR10 coiled
- Lane 10: TNFR10 digested
- Lane 11: TNFR11 coiled
- Lane 12: TNFR11 digested
There was not enough space to put an undigested duplicate of every sample in, but you get the general idea.
- Gel picture
We observe that none of the digestions have worked, that the products are often of different sizes, that there are several bands and that there is no difference between digested and non-digested plasmids. This might of course be due to a non-working enzyme, but still, the ligations clearly need to be redone.
- pGL4.30-SEAP with NdeI
We ran a gel with these digestions too, with coiled and digested versions of the plasmids again.
- Top of the gel
- Lane 1: ladder
- Lane 2: SEAP1 coiled
- Lane 3: SEAP1 digested
- Lane 4: SEAP2 coiled
- Lane 5: SEAP2 digested
- Lane 6: SEAP3 coiled
- Lane 7: SEAP3 digested
- Lane 8: SEAP4 coiled
- Lane 9: SEAP4 digested
- Lane 10: SEAP5 coiled
- Lane 11: SEAP5 digested
- Lane 12: ladder
- Bottom of the gel
- Lane 1: ladder
- Lane 2: SEAP7 coiled
- Lane 3: SEAP7 digested
- Lane 4: SEAP8 coiled
- Lane 5: SEAP8 digested
- Lane 6: SEAP9 coiled
- Lane 7: SEAP9 digested
- Lane 8: SEAP10 coiled
- Lane 9: SEAP10 digested
- Lane 10: SEAP11 coiled
- Lane 11: SEAP11 digested
- Lane 12: ladder
- Note
Wells 4 and 5 on the top and wells 11 and 12 on the bottom of the gel mixed because of power cuts in the lab, it was hard to see anything.
- Gel image
The digestion (or maybe ligation...) doesn't seem to have worked either.
