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Plasmid digestion and gel
We digested the plasmids (LovTAP and RO) of the MaxiPreps made on 12th July.
The amounts of DNA and enzymes where calculated based on the [http://www.neb.com/nebecomm/products/protocol445.asp|NEB Digestion Protocol] which uses 500 ng of DNA.
Knowing we have RO plasmid: 407.2 ng/µl LovTAP plasmid: 294.5 ng/µl
We added
- RO plasmid : 1.23 µl
- LovTAP plasmid : 1.7 µl
Enzymes :
- RO plasmid : EcoRI-HF + PstI 1 µl each
- LovTAP plasmid : EcoRI-HF + XbaI µl each
Expected fragments
- RO : 1472bp + 2255bp
- LovTAP : 574bp + 2642bp
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.
- Digestion time : 2h
- 2 tubes per sample.
- Comments
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