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Digestion
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 more 30 min at 37ºC.
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.
- Comments
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