question for the PCR to cloning construct - (Nov/02/2006 )
Dear everyone:
I am constructing a cloning,but met a problem in the first step.it is no problem to get the right bands using normal Taq ,but when i change to higher fidelity polymerase there are no bands.
can anyone give me any advice?is there anything can optimize?
THANKS
I am constructing a cloning,but met a problem in the first step.it is no problem to get the right bands using normal Taq ,but when i change to higher fidelity polymerase there are no bands.
can anyone give me any advice?is there anything can optimize?
THANKS
Hi reyes,
I think you maight get bend while using higher fidelity polymerase, but you didn't notice that only because of its low amount. I heard that higher fidelity polymerase usually results in low productivity. So maybe you stain your gel for more long while you can see the bend. hehe~
Actually, if I were you, I'll increase the amount of templet and the reaction volumn to do another PCR reaction. Hope the suggestion will be useful.
cheer,
Saltycookie
It happend to me, especially in amplification of sequences from genomic DNA and cDNA, change anther brand of Pfu might be good idea, even the same brand Pfu, some batch work well, some time no.
maybe they are non specific, that is why high fidality doesnt multiply them 
In fact, the normal Taq is more efficient in amplification of target fragments than higher fidelity polymerase.
Taqis more efficient because Taq is insensitive to dUTP. If dUTP is incoperated into the DNA template, Taq will happily go on. High fidelity polymerases stop, try to fix it and may fall off while doing so. dUTP is found at some low level in dNTP mixes but most of it is generated in denova by thermal deamination of dCTP.
Optmise your anealing temperature, your extention time and extention temperature, your Mg ion concentration, you KCl concentration (if you use this), your adjuncts (BSA, glycerol), template quantity (and do check the quality of the DNA is stiill good) and also make sure your polymerase is stiill active and has not died on you while in storage.
Optmise your anealing temperature, your extention time and extention temperature, your Mg ion concentration, you KCl concentration (if you use this), your adjuncts (BSA, glycerol), template quantity (and do check the quality of the DNA is stiill good) and also make sure your polymerase is stiill active and has not died on you while in storage.
Hi Pernese, that's something I had no idea about. Really interesting as I have often tried to understand why templates that ampify with taq don't with pfu. Do you have a source or reference?
at this moment, no. I have to look up the random stacks of papers I have read. Try Googling, it should pull up something on dUTP and Taq.
will do. Thanks.
I had a similar problem before. When I switched to a Pfu-like enzyme I did not take into account the fact that the buffers were different, and consequently so were the TM's for my primer in each respective buffer.