I'm validating if my clone was successful. I first made a colony PCR and the correct band appears. I was then told to validate further using restriction digestion. I added 15 uL of aprox 16 ng/uL concentration of plasmid, 1 uL of BamHI, 1 uL HindIII, 2 uL of Buffer e, and 1 uL of h2o. I digested @ 37c for 1 1/2 hours then ran an agarose gel. My plasmid is 5,899 and the insert is 450 bp. The band after electrophoresis appears @ aprox 2,100 bp. I then ran another gel using 10 uL of 187 ng/uL concentration of plasmid uncut. I got the same band @ 2,100. I'm thinking my clone is supercoiled due to the crescent shape. Did I use too little plasmid in the restriction digestion?? Can an uncut plasmid migrate from 6,500 to 2,100 bp???
You seem to have quite a lot of DNA in the gel pocket. Check if you have determined the concentration of your plasmid correctly. I think that the bright band you see is not supercoiled DNA (you would probably see several weaker bands above the bright band due to DNA nicks and linearization and below the bright band due to circularization). If possible, do a digestion with only one enzyme and ensure that it has the optimal restriction conditions (buffer, temperature, etc.). Check both enzymes if they work correctly. 200 ng of plasmid DNA should be enough for the digestion reactions.
Good luck!
Regards, Christian
@Christian: Did I mention that I extracted the plasmid dna from a BL21 (DE) colony I had. The plate was about 3 months old. Could it be that the plasmid dna has endonuclease contamination, or it got degraded from the mini prep purification process?? The only explination that I can find is that the 2,100 band that is appearing is my degraded clone which contains the fragment (hence why the colony PCR showed positive).
Better to use a fresh clone for plasmid DNA isolation. Maybe you can get a new clone? Try to re-transform the plasmid DNA you have into E. coli competent cells. If you are lucky, some colonies will contain the right plasmid. Then do a fresh preparation.
Hope this helps a bit.
Cheers, Christian
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