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PCR products 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.
Digestion of the LovTAP PCR products with NotI and SpeI.
Digestion of eGFP and TNFR PCR products with HindIII and FseI for insertion into pGL.
Digestion of L2, L3, L5, L9 pMP-LovTAP minipreps with HindIII and XbaI for verification of plasmid quality (figure out what this backbone actually is.
Digestion of pMP for LovTAP insertion with NotI and SpeI.
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).
- Gel top
- Lane 1: ladder
- Lane 2: nothing
- Lane 3: pMP3
- Lane 4: nothing
- Lane 5: pGL
- Lane 6: nothing
- Lane 7: LovTAP2 coiled
- Lane 8: LovTAP2 digested
- Lane 9: LovTAP3 coiled
- Lane 10: LovTAP3 digested
- Lane 11: nothing
- Lane 12: ladder
- Gel bottom
- Lane 1: ladder
- Lane 2: LovTAP5 coiled
- Lane 3: LovTAP5 digested
- Lane 4: LovTAP9 coiled
- Lane 5: LovTAP9 digested
- Lane 6: LovTAP PCR product
- Lane 7: LovTAP PCR product
- Lane 8: GFP PCR product
- Lane 9: GFP PCR product
- Lane 10: TNFR PCR product
- Lane 11: TNFR PCR product
- Lane 12: ladder
- Gel picture
- Comments
- pMP and pGL digestions failed.
- The only PCR products showing up are on the GFP lanes.
- Digestion of minipreps not giving the correct bands.
