Hi,
I want to amplify fragment of approximately 3100 bp from bacterial genome for cloning in pET28a using NcoI and XhoI restriction enzymes. I performed PCR using following program:
Pre-denaturation: 98 oC for 5 min.
Denaturation: 95 oC for 1 min
Annealing: 68 and 70 oC for 1 min
Extension: 72 oC for 6 min
Go to 2- 35 times
Final extension- 72 oC for 10 min
I used Pfu and Taq polymerase enzymes for PCR reaction separately and performed PCR with both Annealing: 68 and 70 oC for each enzyme separately. Tm of primers is 72oC and length of each primers is 31 and 30 bp.
but i didn't get any amplification fragment on 1% agarose gel electroforesis. What might be the reason that I can't get any fragment?
Elnaz Afshari
For amplification of DNA size more than 2kb you can use either Phusion Taq or LA Taq (TaKaRa Biosciences) .
There are the several factors which influence in the amplication of dna size more than 2kb.
1.One of them is the GC content in your amplicon . If it is more than 40% then you should use GC buffers for PCR. Both Phusion as well as LA Taq comes with GC buffers.
2.Another is Tm of the primer, use a primer having Tm more than 60 degree for better result.
3.Extension time with Phusion and LA Taq should not be more than 2 min.
3.Initial denaturation temperature and time play crucial role in these type of pcr reaction.please avoid initial denaturation temperature more than 93 degree for 2-5 minutes for LA Taq while 98 degree 2 minutes for Phusion Taq
4. Remember not to use more than 200ng of total DNA per reaction .
When you say cloning with 2 enzymes are these enzyme recognition sites built into the primers. If they are and also if you have any extra bases at the 5' end of the primers all of these bases must not be included when calculating the annealing temperature. For the first 2 cycles these bases do not exist in your template dna so if your primer is 30 bases but only 24 bases are homologous to your template dna then 24 is the length of primer to calculate annealing temperature
Dear ELnaz
as Sumit suggest for long fragments a better taq cooul be required. Standard Taq is not able to go up to 2-2,5Kb.
Generally for long fragments i'm using KApa Hifi (roche) , Q5 (Neb) or CLone AMP (Takara) which are also high fideliry and guarantee you low risk of mutations.
A question: When you calculated the Tm of your primers (70 and 71°c), did you calculated the Tm only of the region that anneal in the template (with out include the restriction enzime site and the addictional bases at the 5' or did you calculatedthe Tm of the final primers?
ciao
Manuele
I agree with Manuele and Sumit. High fidelity Taq should be used for long fragments. I tried amplifying around 4000-5000 bp from rice for cloning and used Phusion for this purpose. In most cases, we are successful in getting the desired fragment size. We always perform a touchdown step to increase the likelihood of amplifying the product.
Sumit kumar singh Paul Rutland Manuele Martinelli Irish Bagsic- Posada
Hi everyone
Thank you for answering my question
I considered the Tm of the region that include the restriction enzyme site, therefore when i calculated Tm of only 24 bases are homologous to my template DNA and performed PCR again by following program, I saw a band with size of between 2000-3000 bp:
Pre-denaturation: 94oC for 5 min.
Denaturation: 94oC for 1 min
Annealing: 49oC for 1 min
Extension: 68oC for 6 min
Go to 2- 30times
Final extension- 72 oC for 5min
but my desired band is 3100 bp.
what can i do to amplify the exactly 3100bp fragment?
Dear Elnaz
of course design new primers with high Tm ( to perform a pcr with 55-60 annealing ) could be nice to increase the specificity but is it possible that also in this case you need to change polimerase because as all we already told you, the standard taq polimerase is not able to amplify fragments larger that 2-2.5 kb and you need to use an other polimerase as phfusion, kapa, clone amp, pfuultraii , pwo.
manuele
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