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PCR cleanup and insertion of LovTap into psb1-C3
- LovTap Pcr fragment (flanked with BB prefix and suffix) run through Machery Nigel pcr cleanup kit.
- psb1-c3 and LovTap Pcr fragment were digested with ecorI and pstI and ligated
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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