SmaI blunt end ligation problem... - (Apr/05/2011 )
I am stuck with my ligations here. I want some help, whether to try something new or its possible. I have an amplified insert with primers incorporating a 5´-SmaI and a 3´ NotI.
primer Forward 5'- GGG GAG AAG AAC AAG AAT GCC -3'and
primer Reverse 5'- TCA TGC GGC CGC TCA TTT GCT GTT TTC TGT GAA -3'
I did gradient to the template using taq pol, and then using pfu taq amplified fragment of size 2.8 kb as expected. Now this amplified fragment contains internal SmaI site too. So one end was made cohesive with NotI and I am trying to ligate it to a SmaI-NotI cut vector (backbone Size, 3 kb), thinking that other ends would ligate automatically under t4 DNA ligase treatment. I almost have tried a lot (4 times) to with the transformation using the ligates with 1:3, 1:6, and 1:9 vector to insert and also 3 to 4 fold high ligase. But no success, once I got around some 50 to 60 colonies, i selected and colony PCRed 10 but none positive, plasmid isolated upon restriction gave off nothing. I am really being pissed off by the boss. And he is not listening to me to try for new forward primer with any other res site (for stickiness). I cant use slang here but you can understand.... I realy need help.
The 5´ primer starts with GGG, is there a problem with that?
Insert size almost similar to vector, is that a problem?
I am using NEB T4DNA ligase, Qiagen Gel exrtraction kit and not exposing for long the gel slice to uv upon cutting. I am really grateful to you if you could help me out.....
if you have the XmaI available, cut with XmaI instead of SmaI. They both have the same recognition site, but while SmaI leaves blunt ends, XmaI leaves a 4bp overhang, which should be easier to ligate.
XmaI not only produces sticky ends, it also cuts at 37C rather than 25C
The guard sequence around the NotI site is only4bp. This is too short (digest efficiency ~10%).. NotI requires at least 9bp to cut efficiently. As a rule of the thumb most restriction enzymes require 6bp flanking the restriction site. You will need to design another primer.
NEB technical guide is a good place to obtain this kind of information.
Stretches of Gs tend to be bad things in primers. They form Guanine tetraplex structures.. and become less available for template annealing. If possible avoid primers which contain 4G in a row and longer.
THANKyou for your suggestions..... Both the good people...
I tried to increase the ligase on twice SmaI restricted plasmid, it worked. Ya I could have used XmaI for cohesive ends but provides the same site was not there in my insert, which was there. Hooooofff.... It takes 4 out of 20 colonies screened to get this.. for me... and a uL of good luck....
Boss will always be a boss... and never lets holidays... hihihihiih...