cDNA problem - (Oct/30/2006 )
hello every one
i got a problem i want to cloning cDNA from (MDA cell) whene i ligation with hte vector after that
transformation i don't have any colony on the plate so please if any one can help me vector size (5281 bp)
could you give us more details? As many as you can. For example How is the quality of you cDNA? Size? What kind of cloning is this? TA cloning? Conditions that you use. The controls that you have conducted.
There are many places in the protocol that could go wrong. So it is a little difficult to diagnost the problem without further information.
yes you'r right i want to do cloning for cDNA library and the size i used (2000bp) i used with subcloning .
If you're cutting your vector with 1 restriction enzyme (RE) , you have to dephosphorylate your vector first.
If you're cutting your vector with 2 RE or if you're using T/A cloning vector, there's "less" problem.
Gel purify your vector to ensure that uncut plasmids do not contaminate your purified product.
NOTE: RE step, use ~5U of RE per 1 ug of DNA. Incubate at 37C for 1 to 3 hours.
If you're cutting your cDNA with 1 RE or 2 REs, purify it by agarose gel isolation and purification method. I prefer this method. Alternative, you can also PCR purify it (not so good).
Increase insert DNA in the ligation step. Use 100 to 150 ng vector.
For T/A cloning, make sure you're not using proofreading polymerase that'll "blunt" the A overhang.
thank you I love MSGs but how can i know the orientation of the insert if i do TA Cloning
if i do PCR i will lose many of library i want
Sorry for late reply. Took a break from work.
Normally, I'll just do sequencing using universal primers (M13s or T7). Or you can do PCR using a combination of M13 primers and your gene specific primers (Works sometimes, not every time but that'd be less expensive and a bit faster).
<Quote>if i do PCR i will lose many of library i want<Quote>
Are you doing cDNA library construction? Or just isolating your gene of interest?
If you're doing cDNA library construction, it's best to use kit for this purpose.