PCR SOS!

SURESH KUMAR K.G. skg at biochem.iisc.ernet.in
Tue Feb 27 10:49:34 EST 2001


Dear Huang,

You can perform invivo mass excision using total library to convert
lambdaTriplEx2 to pTriplEx2. But since you have the total sequence of your
gene and PCR primers ready, you can directly amplify the gene from the
total library. Conversion to plasmid is not required for this purpose.
You should keep in mind that this direct PCR (from library) will work only
if your gene is represented by a full length cDNA in the library. OR
atleast the seqeunce corresponding to the primers should be present in the
cDNA clone.
If this is not the case, you may try RT-PCR using polyA RNA or total RNA
isolated from tissues expressing your target gene to a reasonably high
level.

Direct expression of proteins from pTriplEx2 cDNA clones never worked for
me, even though the kit says one third of the protein expressed will be in
the correct reading frame. You may consider subcloning your cDNA into
more reliable and suitable expression vectors.=20

Hope this helps.

regards
Suresh Kumar K.G
Department of Biochemistry
Indian Institute of Science
Bangalore-12
India


 On 27 Feb
2001, David Huang wrote:

> I am a customer of the  product K1051-1(Catalog#)(Smart cDNA Library
> Cinstruction Kit, produced by Clontech company). I have established my cD=
NA
> library using his kit and achieved wonderful results. I have got a librar=
y
> in the term of =A6=CBTriplEx2 , but I want to convert the library to the =
term of
> plasmid. I learned from his manual that conversion may be performed on
> individual positive paques picked from the secondary or tretiary screenin=
g
> plates( using  host strain :E.Cloi BM25.8). So  I want to know if the tot=
al
> library can be converted to plasmid term .
>     As you know , I want to get a gene for expression from the library
> constructed according his Kit User Manual. I have total sequence of  my
> target gene and devise my PCR primers. But I don't know how to get my gen=
e
> from the library. Would you mind give me some advices?
> Thnk you very much!
> Your truly:HuangWei
> 2001.2.22
>=20
>=20
> <http://www.biowww.net/forum/read.php?f=3D1&i=3D923&t=3D923>
>=20
>=20


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