I need to clone a 5415 bp gene into pglo plasmid replacing it's GFP gene which is around 700bp long. Now PCR amplification of this 5415 bp gene using PhusionR HiFi Taq doesn't show any band, only smears are seen. I tried many times. Can anyone help providing me useful suggestion?
Depending on the target DNA, PCR amplification of more than 5 Kb is not always easy. If you share some more information, about your template, primers, PCR protocol, you increase your chance to get useful suggestion.
I agree with Uwe. My first guess (without knowing details) would be that you need to redesign your primers because most of the time that's been the issue when I've gotten smears but there can be a lot of reasons.
Dear Nadia
I suggest to you to try other polimerases as
kapa hifi (Roche)
Clone amp (takara)
which are working well in my hand to amplify entire plasmids with size higher or comparable to the one of your insert.
good luck
Manuele
Dear Nadia
I suggest you:
a. get better quality of genomic DNA
b. PCR with template concentration gradient
c. change different enzyme, such as takara primer star MAX.
d. redesign your primer
e. Using temperature gradient annealing PCR or try 2 step pcr protocol
f. if failed above insert 3000 kb first, then 2415
good luck
Thank you everyone. Tell me one thing is their any possibility that 5415 bp can't be inserted in almost same size plasmid (5371bp) ?
Thank you Yongbin Li .
But I predict the expression efficiency will decrease,right?
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