Amplification of cDNA inserts from freeze-thawed phage

[Jorge Vasquez-Kool]

I am trying to amplify cDNA inserts from phage (ZAP cDNA library) to
screen for insert size and to prepare template for sequencing some cDNA
inserts. The plaques were picked with toothpicks (stabbed) and the
toothpicks were put in 96-well blocks containing 200 µl SM buffer and 10
µl chloroform. The toothpicks were removed after 30 min.

So far, I have been using Boehringer Taq and the following reaction
cycle:

(0:20 @ 94°C;  0:30 @ 63°C;  2:30 at 72°C) x 10			
(0:20 @ 94°C;  0:30 @ 56°C;  2:30 at 72°C+1 sec/cycle) x 20		
(5min @ 72°C) x 1;	

and conditions:

freeze-thawed phage	5 ul
10 x BM buffer (1.5mM Mg)1 x
dNTPs			200 µM	
MgCl2			1.0 mM
FORWARD PRIMER		50 ng	(0.38 uM)
REVERSE PRIMER		53 ng	(0.38 uM)
Taq Pol			0.75 U	
Total			25 ul		

The best ampplification rate so far was approx 65% but 50 % is more
typical. Freeze-thawing only improves the rate with 5-10%. Amplification
is great if it amplifies (all-or-nothing situation).

Does anybody have a good reliable method for amplifying inserts from
phage?

My email is:

aamyburg at unity.ncsu.edu