Amplification of cDNA inserts from freeze-thawed phage

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Wed Jan 14 06:13:33 EST 1998


In article <34BBA69C.1D46 at unity.ncsu.edu>, Jorge Vasquez-Kool
<vasquez at unity.ncsu.edu> wrote:

> 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


Hi Jorge,
I´ve often done PCR from phages. I think your problem is the amount of
template DNA. Of course, in theory PCR should work from 1 molecule, but in
practice you need much more. If you remember that 1 plaque contains
approximately 0.25ng of lambda-DNA, it is well possible that you don´t
have enough template molecules in your 5ul SM buffer to get a good
amplification with 20cycles only. I do it in this way:

* prepare your PCR mixture, containing everything but your template DNA.
* use a toothpick and slightly touch a plaque; Then put the toothpick into
50ul of SM buffer. Use this as your phage stock for later use, but do not
use it for PCR!
* Then use a yellow tip, cut away approximately 2mm from the front (so
that you get a kind of wide bore tip).
* use this tool to pick the entire plaque together with the top-agar, but
not the bottom agar.
* blow the plaque into your PCR mix
* start PCR with a program according to your primers and the length of the
fragment to be amplified. I usually use 50C for annealing (30"), then 72C
for extension (30"-2´; roughly 30" per 1kb), then 94C for 30". Do 30-35
cycles.
An initial 5´ at 95C is enough to melt the agar and break the phages.
* analyse 1/10 of the PCR an a gel

Hope this helps,
Frank



More information about the Methods mailing list