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BclI digestion - (Feb/03/2006 )

Hi, is there anyone who has experiences with BclI restriction enzyme?
I have the next promblem: it won't cut my plasmid! I am aware of the special incubation temperature and the methylation blockage. I collected the plasmid from SCS110 cells (Dam-). Sequence analysis validated the right sequence, but still the enzyme won't cut (another plasmid is digested properly, so the enzyme works perfect...). What else can it be?

Please help me!!!!
Thnx

-pjansen-

There might be an inhibitor in your DNA prep. You could test this by mixing a small amount of the DNA you can cut into a sample of the DNA you cannot cut and verify that the mixture still results in a cut of the positive control DNA.

If you have inhibitors, then re-isolate or re-purify your DNA. If you are doing phenol/chloroform extraction, make sure you do a chloroform only extraction as the last step to remove traces of phenol.

-phage434-

QUOTE (phage434 @ Feb 4 2006, 10:18 AM)
There might be an inhibitor in your DNA prep. You could test this by mixing a small amount of the DNA you can cut into a sample of the DNA you cannot cut and verify that the mixture still results in a cut of the positive control DNA.

If you have inhibitors, then re-isolate or re-purify your DNA. If you are doing phenol/chloroform extraction, make sure you do a chloroform only extraction as the last step to remove traces of phenol.


I checked it, and there appears to be no inhibitory effects, because in the mixing digestion, only the positive control ia cut.

Any other suggestions?

-pjansen-

in order to be sure the strain is dam-, you may digest with an other enzyme sensitive to dam methylation.

-fred_33-

QUOTE (fred_33 @ Feb 6 2006, 07:57 AM)
in order to be sure the strain is dam-, you may digest with an other enzyme sensitive to dam methylation.


I have already checked that, and that wasn't the problem.
I have made my plasmid again, but now I contruscted the plasmid in Dam+ cells first, then transform the isolated DNA to Dam- cells. Now the BclI digestion gives no problems. Although I am very happy now, I still would like to know why it appears essential to create a plasmid in normal E coli cells in stead of dam- E coli cells.

unsure.gif

-pjansen-