Not sure with cloning - (Jan/14/2007 )

HI,

I always had problems with cloning.
I have few questions .
Is there any way to chk that the LB (ampicillin) plate I prepared is working?
How long plates and compitent cells can be stored?
I have one vail of 50 mirol compitent cell left , and I also have a little bit of topo vector with me . I have to clone two genes for sequencing both the size are ~200bp, besides I also have new promega ligation kit . Topo has always been easy to use. But I am not sure about the promega .

So what can I do with the options available .

Is it worth going for ditrect sequencing?? what are the things required for that . I had a bad experience once. my primers were not good enough.

Please help

regards
vani

i think i am not sure what is your question.

yes you can store electrocomp cells at -80 in eliquots if they are in glycerol.
you can test your plate by adding just electrocomp e-coli to it. nothing grows, your antibiotics are fine.

have no experience in sequencing or topo vector sorry.

Is there any way to chk that the LB (ampicillin) plate I prepared is working?
spread some untransformed e-coli on the plate, if nothing grows, the antibiotics are OK.

How long plates and compitent cells can be stored?
plates, i've never had the around long enough to find out. cells, at -80'C for a long long time.

I have one vail of 50 mirol compitent cell left , and I also have a little bit of topo vector with me . I have to clone two genes for sequencing both the size are ~200bp, besides I also have new promega ligation kit . Topo has always been easy to use. But I am not sure about the promega .

So what can I do with the options available .

1. order more ecoli. 2 grow up more e.coli. there are a few protocols available, and ways to test their competency. topo is supposed to be easy to use. promega, never used it.

direct sequencing??
i think it really depends on the size of the thing you want to sequence. as well as DNA quality, etc. i think that cloning and sequenicng is the best bet (and unfortunately more time consming).

V