Cloning Problem - (Feb/23/2006 )
I have did a one mistake and I have now problem for gettig clone.
I have to clone my insert into pET32 and I make my Primer with ECOR5 and ECOR R1.
Both these sites are very much near and separated by just BAM H1 sites and I am now not gettin clone. I think its just because of restriction digestion because sites are very close to each other.
Is it possible to do more work on it or
just cut the vector with ECO R5 and leave the inset uncut and try for both end blunt cloning.
I am waiting or your suggestions.
if you are cloning into a pET vector, do you not wish to express the protein later? if you change your strategy, will it result in a frameshift and make you unable to achieve expression? if your goal is merely to get your insert into a plasmid, I do not see why it wouldn't work (you will probably get the insert in both orientations, though). but, if you want protein expression, there may be a problem with your method.
does this help?
does this help?
But suggest me is it possible to cut vector with ECR R1 AND ECRO RV.
Both sides are very much near and just separated by Bam HI.
Is it will cut by enyzme or not either in double digest or single digest
did you design your insert by PCR? how many bases did you leave at the ends? maybe it is not cut from the first place? i mean maybe close sites are not the problem....
cut your vector pet 32 with ecorv only and ligate your fragement in this vector but if u want to later cut and other characters u want to make it is not possible ok
otherwise if u r going to express u r protein? if yes u choose another pet vector or some expression vector which having 2 restriction enz sites(your 1st and 2nd restrn enz) at different places or u go for vector having only one restriction site(either 1st or 2nd enz) then u cut it and paste u r fragment and express it.
if ur not going to express u choose cloning vector of above mentioned charcteristics
I keep 6 bp of AAAAAA before enyzme site. But I have no clone get yet. I am making PCR with pfu polymerase. It wll make no overhung but.is it or not.