I want to over lap two DNA sequence
A : 378 bps
B : 374 bps
Taq PCR protocol :
A sequence template 1ul (600pg/ul)
B sequence template 1ul (600pg/ul)
Foward primer 1ul (10mM/ul)
Reverse primer 1ul (10mM/ul)
10X Taq buffer 5 ul
ddH2O 39ul
---------------------------------------------------------
95 °C heat up 10min
on ice 5min
---------------------------------------------------------
Taq 1ul
dNTP 1ul
----------------------------------------------------------
anneal temperature 55°C 35sec
extension 72°C 50sec (1kb/min)
With this protocol I got the full length template (~750bps) but I need to use Pfu to transform
Pfu PCR protocol :
A+B sequence template 1ul (600pg/ul)
Foward primer 1ul (10mM/ul)
Reverse primer 1ul (10mM/ul)
10X Pfu buffer 5 ul
ddH2O 40ul
---------------------------------------------------------
95 °C heat up 10min
on ice 5min
---------------------------------------------------------
Pfu 1ul
dNTP 1ul
----------------------------------------------------------
anneal temperature 55°C 35sec
extension 68°C 2min (500bps/min)
I have changed annealing temperature from 57°C~47°C per 2°C;
however I can only got template lower than 500 bps in any case
Pfu DNA polymerase has superior thermostability and proofreading properties compared to Taq DNA polymerase. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that as the DNA is assembled from the 5' end to 3' end, the exonuclease activity immediately removes nucleotides misincorporated at the 3' end of the growing DNA strand. Consequently, Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts.
So raise your temp and time of extension step.
Regards.
Alireza Mordadi
You mean I need to raise my annealing temp and extension time ?
But as my primer design, Tm is about 60°C and I had tried annealing temp at 57 °C but still not work.
As for extension time , the commercial protocol said that Pfu can produce 500bps/min; however I have already set 2min , isn't it enough time in my case ?
Taq is a highly active, but relatively poor fidelity DNA polymerase. Pfu is high fidelity, but rather poorly active enzyme. You may try other enzymes that combine high fidelity with high activity, such as Phusion or Q5. I don't see why you should use Pfu to transform, except because of its high fidelity. You could also use Taq, but in that case, be sure to check several clones by sequencing to verify the absence of spurious mutations
My meant is extension time and extension temp, annealing temp is good regarding to performed by gradient.
Also consider nice advice of dear Pierre.
Regards
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