double digest not working? - BamHI+SacI double digest (Aug/12/2005 )
Hi All,
i'm trying to put in an adaptor into vectordigested with BamHI and SacI, i did not dephos my vector and phos oligos, in first set, i digested with BamHI, gel purified, then digested with SacI, gel purified and ligated, i got colonies, got them sequenced but no insert was there, only EcoRI site b/w these two sites as in original vector. then i reversed the order of digestion, and ligated only vector, no insert and got colonies, but got no colonies in vector only ligation of my previous preaparation, i don't know what to do, in all probability one enzyme not cutting, but both have high end-cutting efficiency, please help me get past/resolve thi sproblem.
thanks
nbj
The problem you have is probably caused by the second enzyme not cutting efficiently when close to the end of the linearised vector. This problem is compounded by the fact that you don't have any difference is size between the vector cut with two RE and when cut with only one RE.
What you really need to do is SAP treat your vector and PNK treat your oligos. If you are clever you can SAP treat only one RE site and PNK only one of the oligos to avoid getting concatemer inserts.
Daniel
Improved ABI3730 DNA sequencing traces
i'm trying to put in an adaptor into vectordigested with BamHI and SacI, i did not dephos my vector and phos oligos, in first set, i digested with BamHI, gel purified, then digested with SacI, gel purified and ligated, i got colonies, got them sequenced but no insert was there, only EcoRI site b/w these two sites as in original vector. then i reversed the order of digestion, and ligated only vector, no insert and got colonies, but got no colonies in vector only ligation of my previous preaparation, i don't know what to do, in all probability one enzyme not cutting, but both have high end-cutting efficiency, please help me get past/resolve thi sproblem.
thanks
nbj
hi,
thanx for suggestion, but i've no idea how to phos/dephos only one site, also i'm not sure if my insert which is ds linker/adpator i made following annealing of ss oligos, is annealed correctly ( i run gel, can't say for sure, i used some of annealing protocols discussed in this forum), following dephos of vec, if i ligate only vec, no colonies but when i ligate with insert, get colonies without insert, got any clues ??
thanks again
nbj
What you really need to do is SAP treat your vector and PNK treat your oligos. If you are clever you can SAP treat only one RE site and PNK only one of the oligos to avoid getting concatemer inserts.
Daniel
Improved ABI3730 DNA sequencing traces
I think Daniel is correct, as well, your sites may be to close.
I would order phosphorylated oligos to avoid going crazy and as far as annealing your oligos for the adapter, try differing concentrations of your oligos and use a simple method for annealing. Bring the mixture up to 95-100C for 5-10 min in a water bath or thermocylcer, remove from heat and let cool to RT on your benchtop.
What you really need to do is SAP treat your vector and PNK treat your oligos. If you are clever you can SAP treat only one RE site and PNK only one of the oligos to avoid getting concatemer inserts.
Daniel
Improved ABI3730 DNA sequencing traces
I think Daniel is correct, as well, your sites may be to close.
I would order phosphorylated oligos to avoid going crazy and as far as annealing your oligos for the adapter, try differing concentrations of your oligos and use a simple method for annealing. Bring the mixture up to 95-100C for 5-10 min in a water bath or thermocylcer, remove from heat and let cool to RT on your benchtop.
hi,
maybe u wanna try topo cloning...using the invitrogen kit. this worked for me. i did an experiment in which the double digest didn;t work. so i then did topo cloning and then did double digest and it worked. u could try. ask your supervisor...but, actually i am new to this field. i dun quite understand how topo cloning or TA cloning helps to solve the problem. anyone has any idea? thanks.