help lambda-ZAPII-PCR??

K.D.Preuss tm12kpkp at rz.uni-sb.de
Tue Jun 21 07:44:32 EST 1994


Hi,

we are trying to amplify lamda-ZAPII phages directly after picking the phages
from a cDNA library. We are checking the method by randomly picking a single
plaque, elute it in 100 ul SM and making PCR by using M13forward/M13reverse
primers or the T3/T7 primers. We failed in getting an insert. We are trying
the same procedure with a purified phage clone and primers specific for the
insert. Same result. We tried it with more ul of phage and we played
around with Mg2+ concentration and annealing temperature down to 40C.
We also failed.
But after doing invitro excision with the ExAssist system the PCR is working
well with all the primers we tested.

We want to use this PCR screening method to look for relatively rare clones
in our library. In priciple we want to follow the protocol which is
published in BioTechniques 16:420-422 (1994) by Lardelli.

Any sugestions why the phage PCR isn't working with lambda-ZAPII?

Please E-mail to tm12kpkp at rz.uni-sb.de


Thanks
Dieter
K.D.Preuss                      E-MAIL: tm12kpkp at rz.uni-sb.de
Physiologie II                  Phone: +6841-166123
Universitaet d. Saarlandes      FAX: +6841-166655
66421 Homburg/Saar
Germany



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