Dear all, I am currently working on antimicrobial mechanisms of Campylobacter and i want to ascertain whether tetO gene were located in the chromosome within the CJSA_0191 locus or located on pTet plasmid. Thus i used 2 primers, one used to generate an
amplicon size of approximately 2000bp and it was successfully identified and the other one designed based on the pTet sequence were employed for an amplicon size of about 3500bp and i tried many times to amplify this gene but failed to do so. please guide me.
Hi,
3,5Kb should not be an issue for PCR mixes. The quesiton is if your primers are working niely together. In addition you would need to increase extension time but in general it works. In addition check your DNA quality as highly degraded DNA might prevent the amplificaiton here
Sven
Hello,
I agree with Sven Reister. The amplification of 3.5kb sequence should not be any problem for standard DNA polymerase. Generaly for longer amplification a selected enzymes are used (you can have a look on previous posts on RG) but not in your case. Remember about the extension time - I think that 2.0 - 2.5 min will be enough to get your products.
Good luck
Piotr
As others have said, 3.5 kb is a reasonable size product for most PCR enzymes. Check that your annealing temp is optimal, increase the extension step time, and you might want to add DMSO if you have a high GC content (if you do add DMSO, then you'll need to also lower the annealing temp).
Amal Awad In case this is interesting just write me a message and I can give you more details regarding suitable chemistries and optimization approaches
Hi Amal, it is a fact that PCR amplification for the products above 3 kbp are often compromised with non-specific primer annealing mainly due to secondary structures present within the template. And, whenever in a PCR there is no product at end, so multiple factors must be under consideration, as may be our primers are not good design wise, their Ta is not appropriate, may be number of cycles are not enough, quality and quantity of template also matters badly, template GC content, type of polymerase used in PCR etc.
Elongation time, in a normal PCR as standard is kept 1 minute for 1 kb extension as a rule of thumb, but is dependent on the use of polymerase type and your final product size.
For products less than 10 kb, extension temperature is normally kept at 72 C and primer concentration should be less than 0.5 uM (equivalent to 0.5 pmoles / uL or 500 nM) and less than 25 oligomer primer size is quite fine.
Remember Taq polymerase works for more or less 5 kb effectively, but it is best for 3 kb product and having an extension rate 50 sec / kb, even not a minute. On the other hand, it has low performance for GC rich templates. So in such case, you can look into other available polymerases, as may be they can solve your issue, like the high fidelity (HF) ones for example; Phusion, DeepVent, Q5, KAPA2G etc.
Regards...
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