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Digestion of PCR products
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.
Purified PCR products (three melanopsin readouts) from the gel from the previous day were used. They were digested with the enzymes that would allow for a ligation into the pGL4.30 plasmid. pGL4.30 contains the NFAT response element that is necessary for the melanopsin calcium cascade.
We knew, though, that the Nanodrop DNA concentration results from the gel purification were extremely low, around 5 ng/µl. Feedback from other people showed they had already had problems with the DNA concentration after use of the QUIAGEN kit. We tried re-washing it with the PE buffer and re-eluting, but the yield was still extremely low.
As these concentration problems had no apparent downstream effects, according to the internet, we decided to proceed further.
Remark: GFP and TNFR use the same backbone digestion (and, logically, have the same restriction sites added to them), and SEAP has a different one because of a restriction site inside of the gene.
- Digestion mixes
- GFP with FseI & HindIII:
- GFP purified DNA: 16 µl
- FseI: 2 µl
- HindIII: 1 µl
- 10x BSA: 5 µl
- 10x N2 buffer: 5 µl
- H2O: 21 µl
- TNFR with FseI & HindIII:
- TNFR purified DNA: 16 µl
- FseI: 2 µl
- HindIII: 1 µl
- 10x BSA: 5 µl
- 10x N2 buffer: 5 µl
- H2O: 21 µl
- SEAP with MfeI & HindIII:
- SEAP purified DNA: 16 µl
- MfeI: 1 µl
- HindIII: 1 µl
- 10x N2 buffer: 5 µl
- H2O: 27 µl
The mixes were incubated for two hours, then stored at -20°C for ligation later on.
