Problem with Genbank screening - please help... (Jul/26/2006 )

hallo,
i have a problem with my the screenen of my Cosmid Genbank. i screen it with PCR. i used 3 Primer pairs and i always get a weak signal with the expected size by all cosmids! sometimes i can recognize the positves (signal is intensiver), but it makes the screening not reliable...have someone explanation for that??

thanks in advance
orwah, Germany

N.B: the negative control, which contains no DNA, shows no signal!!

You doubt the cosmid sequence from GenBank(NCBI etc), don't you?

But I think there is no problem with sequence itselt, because you got the right bands, anyway.

I don't know how you prepared your template, but you should remember cosmid has one-copy-number. Possibly you did not put your template enough in your PCR mix.

Why don't you extract your cosmid and treat with RE? Cosmid prep is harder than plasmid prep, but anyway, it's possible. Use 10ml E.coli culture for one prep.

Good luck, anyway.

thanks a lot yja97!

i made my cosmid library with SuperCosI. then i packed the cosmids in Gigapack (stratagen) and transformed them in E.coli sure . i picked 3500 colonies and then i saved them in 96 microtiter plates with Lb and the right Antibiotic.

then from every 96 cosmid-microtiter plates i made 2 Lb with the right antibiotc (48+48 colonies). i let them grow for 20 hours and then i washed them and extracted the cosmid through minipreps but with 3 fold of volumes (from the alka solutions I II and III)

from the genomic DNA, i could get a primer pair that amplifies a piece of the right gen, which i am looking for.

now i use the primer to screen the cosmid library, but it does not work well! with the genomic DNA i works good but not with the cosmids! in addition to that, i get that weak band with all cosmids but it can not be compared to the right one from the genomic DNA (the genomic is intensiver).

i think the amount of the template is enough...i use the same amount from the genomic DNA...

anyway thank u very much and i hope that i could explain my problem now...

has anyone an idea..??

If you used column in the preparation, I still think the amount of the template is not enough.
Column preparation is eligible for the DNA upto 10kb, but cosmids libraries have larger DNA, right?

Of course, there are possibilities of false positive, but if you use 3 pairs of primer with right bands, I think the possilibility is very low.

Anyway, that's a very rapid reply.