chimeric cDNA clones : HELP

Gregor Bucher st2153 at zi.biologie.uni-muenchen.de
Wed Nov 17 11:21:59 EST 1999


Hi Jacob!

Don´t know what kind of chimeras you get - do you have different species
in your DNA template pool? Or are the chimeras with genes of the same
species that are conserved in a region? In these cases it could be the
renown "jumping PCR": if in one round the elongation of a product is not
complete and stops in a highly conserved region this product can serve as
a primer in the next round. It could prime on the "right" template (beeing
elongated finaly) or on another template that is similar in sequence. A
member of our lab got that cloning a piece of DNA of a pool of species.
She got chiemeras that start with species A right to a highly conserved
strech and from there on was the sequence of species B.

If your problem is similar than it is the PCR that has to be blamed. they
say that 10% up to 20% may be chimeric. Don´t know how to avoid it. I
would choose annealing temperatures as high as possible, elongation times
not too short and not too many rounds of amplification (max 25) (and no
reamplification!)

maybe this helps,

Gregor


In article <80qe92$htl$1 at carroll.library.ucla.edu>, bobby at ucla.edu (dgdsg)
wrote:

> Hi.
> 
> I clone 16S rRNA genes and it gives a lot of chimeric clones :
> 
> Isolation of RNA with a RNAwiz kit from Ambion, DNase treatment, RT-PCR, PCR 
> and clone that. 
> 
> Any tricks out there??????
> 
> Thanks.
> 
> Jacob Hofman
-- 
Gregor Bucher, Zoologisches Institut der LMU München
0049/89/5902/444
st2153 at zi.biologie.uni-muenchen.de
----------------------------------




More information about the Methods mailing list