Rhodamine dUTP and capillary eletrophoresis

[Jose de las Heras]

"Pragnya Das" <drpragnyadas from gmail.com> wrote in message 
news:mailman.942.1197666010.23109.methods from net.bio.net...
> Hi everyone
> I'm facing a serious cloning issue.
>
> I'm trying to clone a gene into an RNAi vector (pRFP ~6kb), by PCR and 
> then
> subclone it into RCAS (~11kb) vector. Even after cutting out the insert 
> from
> RFP, followed by gel purification, i'm still getting the insert cloned 
> into
> RFP, although I ligate my insert in RCAS. Why is this happening? has 
> anyone
> faced a problem like this before?
>
> Pragnya Das

I think it's more likely that you you are not recloning into pRFP, but 
rather you are picking up background clones derived from a few molecules of 
the original plasmid that was not cut in the first place. Even if you gel 
purify there will always be a few other fragments carried over, as well as 
the band you are cutting out.
Are you recovering many or just a few colonies?
How big is the insert?

If you're only picking up these, it suggests a severe problem with your 
cloning. I assume your digest looks good.
Now wanting to insult you... but I've done this myself: are you sure you are 
using the correct antibiotic? if both plasmids use a different selection, 
and you're inadvertently plating your ligations with the antibiotic needed 
for pRFP, then you'd get a result like you're describing.

If that is ok, are you sure your ligation has worked? Do you have a positive 
control?

Jose