cloning of 1 kb fragment in 14 kb vector - (Jul/26/2007 )
Hi!
I desperately need some help here, I am trying for 2 month now to ligate a 1,2 kb fragment in a 14 kb vector. I am cutting the fragment and the vector with ClaI and XhoI, and also dephosphorylate the vector. The restriction works (checked on gel), I clean everything up with a qiagen kit and ligate 3:1 molar ratio 3 hours at RT. I used different bacterial strains DH5, STLB3, sure, XL1 blue. I even let them grow at room temperature. Every time I get a few colonies on the control plate and a lot more on the "real" plate, but then when I am doing miniprep and digest it with the same enzymes I get a completly different DNA, it is not even the vector anymore but looks complete different. Has anybody any suggestions? I am really desperate. Thanks in advance!
ok
if your insert come out from a PCR, maybe your analyzed DNA is that vector ? try to digest your template by 2 enzymes before having it as a template, and decrease amount of template by 2.
After that, for the vector try this protocol tha works better for me if it's a polylinker you want to digest :
i do 2 30µl-digestions in 2 tubes of 2µg plasmidwith the different enzymes.
Than i pull the tubes and add 0.5µl of each enzyme and go again for 3H.
I dephoshphorylate and column purify.
The idea background, is that there may be steric disturbing by enzymes. So the overnight digestion cut maybe 99% of 1st site, leaving the MCS alone for second enzme action.
if your insert come out from a PCR, maybe your analyzed DNA is that vector ? try to digest your template by 2 enzymes before having it as a template, and decrease amount of template by 2.
After that, for the vector try this protocol tha works better for me if it's a polylinker you want to digest :
i do 2 30µl-digestions in 2 tubes of 2µg plasmidwith the different enzymes.
Than i pull the tubes and add 0.5µl of each enzyme and go again for 3H.
I dephoshphorylate and column purify.
The idea background, is that there may be steric disturbing by enzymes. So the overnight digestion cut maybe 99% of 1st site, leaving the MCS alone for second enzme action.
Hi fred can you explain me about this double digestion more clearly. How long will you incubate your first reaction with single enzymes?
the idea is to separate the first cut of the plasmid. so i do overnight for first digestion.
I do enzyme 1 + DNA in tube 1
enzyme2 + DNA in tube 2
overnight at 37°
mix tubes 1+2, re add small amount of enzyme and go for at least 2H.
I do it for 4h generally, and column purification.
i elute in 30µl.
Then take the OD and go for your ligation.