Alternative to cloning PCR-fragment ...RESULTS

Trond Erik Vee Aune trondaun at chembio.NOSPAM.ntnu.no
Mon Jun 17 03:21:59 EST 2002



Trond Erik Vee Aune wrote:

> Dear colleagues,
> 
> I've been having trouble succeeding in cloning a PCR library (made with 
> error-prone PCR) into my screening vector. I made the primers so that it 
> would be possible to cut the PCR library, as well as the vector, and 
> clone it in. Unfortunately I lose a lot of my library during the cloning 
> and end up with a much smaller vector library than needed.
> 
> Yesterday I thought of an alternative way to establish the PCR library 
> in my screening vector without actually doing any restriction cloning. I 
> thought that maybe I could use the PCR library (the mutated fragments 
> are around 1 kb) as new primers with the screening vector as template in 
> a new PCR. In theory the mutated fragments should anneal to the vector 
> and then the polymerase will complete the strand. After the reaction I 
> could degrade the maternal vector with DpnI (same principle as 
> Stratagene's QuikChange mutagenesis kit) before transforming the 
> complete vector into my strain.
> 
> Some problems I immediately see:
> 
> * The vector is large (8.1 kb) so the polymerase would have to 
> synthezise around 7 kb, is this too much for Taq, Pfu or Expand high 
> fidelity? Any others suitable for such a task?
> * The primers (at ca 1 kb) could anneal to different parts of the 
> vector? Is this likely?
> * The annealing of the primer could be difficult, what annealing 
> temperature should be used?


I tried the method with this PCR set up:

1) 94, 4 min
2) Hold at 87, added pfu
3) 97, 30 sec
4) 50-60, 30 sec
5) 72, 4 min 30 sec (I used a smaller template)
6) repeat steps 3-5 17 times
7) 72, 10 min

I had three parallells which were identical, but had different annealing 
temperature (50, 55 and 60). I used a negative control without primer.

I then added 10 units DpnI and incubated at 37 degrees for over 2 hours.

The results were as follows:
Positive control (1 mikrol LITMUS28 transformed into my competent 
DH5alpha)                        :  carpet
Negative control (only template) :  1 colony
Reaction 1 (50 degrees annealing): 10 colonies
Reaction 2 (55 degrees)          :  8 colonies
Reaction 3 (60 degrees)          :  4 colonies

It seems like it has worked, but the efficiency is way too low. I need 
at least 2000 colonies.

I am thinking about doing some small chenges and trying again. My ideas 
are to change to the following PCR set up:

1) 94, 4 min
2) Hold at 87, added pfu
3) 97, 45 sec
4) 50-60, 30 sec
5) 72, 4 min 30 sec (I used a smaller template)
6) repeat steps 3-5 35 times
7) 72, 10 min

With this set up I have a longer denaturation time and more cycles.

Do you guys have any idea to what can be done to optimize the reaction?

Regards,
Trond Erik Vee Aune







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