Transformation of a 21kb vector - (Mar/19/2006 )
I desperately try to transform a 21kb large vector into E.coli. Well, the trouble is, that I get some clones on the plate, i use to identify them by restriction analysis after mini prep. The Amount of clones is pretty low and I used to transform either heatshock and electroporation.
Now my big trouble is, that after restriction analysis, a big amount of the vector is missing and in most cases, the part of the vector with the origin just is intact. The rest is missing.
So I think, that ligation itself is succesful. The problem is, that i have to prevent E.coli from kicking out the fragments of my vector.
Big Big Thanks in advance!!!!!
i don't get well how you're saying your ligation is well done. Assuming your vector enters the bacteria ligated, E. coli can't kick out either your fragment or the vector asuming you've done a good selection pressure.
hmm ok, you are right. But even when i assume only religands, how is it possible, that the restriction analysis shows only some or even only one band, which size is about 6 -7 kb?
And concerning the selection pressure; how can i adjust it to a sensible factor? lowering the incubation temperature and prolonging time of regeneration?
how long is your vector only? and te insert? if you see one band, i assume it's only your vector self ligated and digested once...
by mentionning antibiotic, the concentration cannot help to have positive clones... (but i remeber that someone in this forum told sthg related to that point... ) i just meant that there was no mistake in antibiotic (i must admit that once, i plate on kana instead of amp (wrong labell of plate ) and i was surprising myself by all contaminations
What kind of vector are you using, does it have repeat sequences or so? (E. coli doesn't like repeats like retroviral LTR's for instance...).
my vector consits of the pUC 18 region and 15 kb are part of SSV1, which transfects Sulfolobus solfataricus ( Archaea). So it constitutes a transformation system for a hyperthermophile
micro organism. My problem is, that a have to check my ligated vector with the desired insert, before i can transform into sulfolobus. But It emerges, that all my e.coli clones recombinated and kicked most of the vector parts out
which stains do you use?
you have to check presence of recA gene.
BL21(DE3)pLysS : F–, ompT, hsdSB (rB–,mB–), dcm, gal, λ(DE3), pLysS, Cmr.
Jm109 genotype : endA1, recA1, gyrA96, thi, hsdR17 (rk–, mk+), relA1, supE44, λ–, Δ(lac-proAB), [F´, traD36, proAB, lacIqZΔM15], lDE3.
I used to try Top10 F' which gave no results and DH5 alpha, where recombination occurs.
I also use Stbl 4, where recombination can't occur and which are suitable for large vectors.
You might consider the pSCANS vector (google) or the commercial version (Copycontrol, Epicentre, epibio.com). This vector propagates at low copy number unless the pUC18 origin is induced. It was developed for propagating large unstable inserts for sequencing.
did you finally manage transforming the big vector into e.coli. I do have also problems getting my vector in e.coli.