digestion with BamHI - SacI - molecular biology (Feb/15/2011 )
hi all, first time on this forum ...
I have trouble digesting with BamHI and SacI. I'm using pGEMT easy vector in the same I put an insert of 1.7 Kb, and now I must release this insert to put it in a new vector. In the insert add cleavage sites for the enzymes mentioned above ...
I try to do a double digestion, but only get to linearize the plasmid, thinking that one of the 2 enzymes did not work, I made two separate digestions, and both look that open plasmid and then purified to change the buffer and made a new digestion with the other enzyme, in the gel i saw a band of 3kb, pGEMT only guess, but there was no insert.
I make the first digestion with a plasmid concentration of 100ng/ul and digestion was make at 37 ° C for 4 hours with 10 units of enzyme.
question ... the insert it's not seen, by the amount of plasmid or low cutting efficiency?? how I can improve this last?? or other possible causes that is not considering???
from already thank you very much.
SacI can be tricky since it is inhibited by salt concentrations >10mM ...so if you plasmid prep is not 100% cleaned efficiently or you eluted in the wrong buffer SacI can be a real pain in the neck. Additionally, SacI poorly cuts supercoiled DNA. You can find more info here.
100 ng for a digest is not that much ...i would use more and since you are trying to release your insert and purify it you should use much more (~3 µg DNA) ...i would also suggest that you do your digest in a larger volume so that residual salts get diluted and do not harm your digest. Consider an overall volume of 100 µl with 2.5-3 µg of DNA.
I first want to thank you for your response ...
I use SacI for the second digestion, first I make a digestion with BamHI and then through a gel I see only the linearized plasmid, then purified, not from gel, using a commercial kit to change the buffer, I'm using a Promega SacI with Multicore buffer and a fermentas BamHI with buffer tango. this buffers can be the problem??
the amount of plasmid was wrong, I'm using 2ug by digestion, 100ng/ul was the concentration. I'll try the volume that you recommended in the case that some salts pass during the purification. I'll tell you later ...
I hope resolved your problems.
SacI cut hardly linear DNA, it takes around 12h if you want to be sure you cut well.
It is better if you start cutting your vector with sacI when it is circular, and then you cut with BamHI, but no longer than 1 hour because of star activity!
And for your PCR insert, cut the sacI site over night.
It helps a lot to clone such fragments.
Thank you very much!!!
I must say that in the end I could cut my vector doing exactly what you suggested, but that I did by trial and fail without knowing which was the reason, now I understood ... je
I appreciate your response, you and the rest of the forum helped me a lot ...
you are welcome!