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Contents |
Digestion
Protocol: Restriction site digestion
- Look for the best pair of restriction sites, ideally with similar digestion temperatures and times.
- [http://tools.neb.com/NEBcutter2/ NEBcutter] for finding cutting enzymes.
- [http://www.neb.com/nebecomm/DoubleDigestCalculator.asp 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.
Enzymes chosen: EcoRI and PstI. (check using NEBcutter online tool) Digestion temperature: 37ºC. Buffer: EcorI + BSA (both parameters using NEB double digest finder online tool)
Each sample (total of 5) will have a total of 50 µl:
- Water: 38.5 µl
- Buffer EcorI 10x: 5 µl
- BSA 100x: 0.5 µl
- DNA (RO plasmid from 29 June miniprep): 5 µl
- EcorI 20 units/µl: 0.5 µl (10 units)
- PstI 20 units/µl: 0.5 µl (10 units)
To save time and tips, we mix all ingredients except #4 (DNA) in a 1.5 µl tube:
- Get BSA, Buffer EcorI and DNA (29 June miniprep) from the freezer and let defreeze.
- In a 1.5 µl tube, add:
- Water: 192.5 µl
- Buffer EcorI 10x: 25 µl
- BSA 100x: 2.5 µl
- EcorI: 2.5 µl
- PstI: 2.5 µl
Total of 225 µl, or 45 µl per each of the reactions.
Note: Enzymes are very sensitive: Don't let defreeze (don't touch the bottom of the tube!). Use them fast and back to the freezer. Don't vortex.
- Distribute this solution in 5 tubes (45 µl per tube).
- Add 5 µl from each DNA sample to the respective tube (then back to freezer, together with BSA and Buffer).
- Keep for 2 hours at 37ºC in the Thermomixer (at 600 min-1).
Since the gel was not yet ready, they stayed 30 more min at 37ºC.
Gel electrophoresis
- Recovered the gel prepared, and not used, the 29 June.
- Microwaved at 7 untill disolved.
- Poured over small box. Let 45 minutes to settle down.
- New ladder solution prepared.
- Added 10 µl of 6x loading dye to each 50 µl digested DNA sample.
- After gel is solid, moved to a small electrophoresis chamber.
- First well: 30 µl of ladder. Then samples (60 µl) 1 to 5, in order.
- Run 40 min at 90 V. Dye has moved only 20% of the gel length.
- Set to 105 V for extra 45 min.
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).
