Hi, is it alright to use an extension temperature of 68 degrees with GXL enzyme for PCR products below 4kb? Takara (GXL's manufacturer) recommends an extension temperature of 72 degrees with GXL enzyme for PCR products below 4kb, and an extension temperature of 68 degrees for PCR products above 4kb. However, I am able to obtain PCR products using 68 degrees extension temperature for PCR products 2kb and above and no products if I use 72 degrees extension temperature for the same PCR products.
thank you.
Since the manufacturer recommends both temperatures then I would be happy using any temperature between 68 and 72 that produces a good product.It is a bit surprising that both temperatures do not work for you and your template may have some secondary structure issues so if your enzyme comes with a high GC buffer that might be worth a try. It might be worth checking the primer and template sequence looking for mismatches under the primer just in case your cycle is not allowing good primer annealing before the extension step.
apologies if I was not clear. I am able to get PCR products at 68 degrees extension temp but not at 72 degrees extension temp for products that are 2kb and above. I am wondering if using 68 degrees extension temp might make any difference to the quality of the products that are shorter than 4kb.
I would not expect that getting a correct size amplimer at 68 would cause any problem with amplimer quality especially as the manufacturer is happy that both extension temperatures are usable
For a single polymerase there are more errors incorporating bases at lower than optimal temperatures with lower temperature . Two factors are important in this case
1 the enzyme extends even at the annealing temperature often at hundreds of bases a second so it is not just the extension step that leads to errors.
Another factor is that many polymerases incorporate a proof reading enzyme or have proof reading ability so the lower annealing and extension temperatures do not lead to incorporation errors as often as expected
suppose I understand you correctly, using an extension temp of 68 degrees (the temp in the product manual) for an enzyme with a proofreading ability such as GXL is fine. It should not introduce more incorporation errors than using an extension temp of 72.
I successfully got the PCR bands on the gel using 68 degrees rather than using 72, and I wonder why. thank you.
You would have to check the error rate of each temperature with Takara but as both temperatures are recommended by Takara I would not expect much difference in the error rate.
I do not know why you can get a product at 68 but no product at 72 but my guess would be that there is some sequence or secondary structure that causes the enzyme to fall off when extending at high speed at 72 but at a lower speed perhaps the enzyme can read through that region. Again checking with Tech Support at Takara might be interesting. It might be worth trying a different buffer at 72 although Takara do claim that this enzyme is particularly good for amplifying high GC and high AT rich sequences
unfortunately, the lab did not accept the manufacturer's protocol and wanted me to use 72 extension temperature as well as slight variations of denaturation time, annealing time, and amount of dNTP from the manufacturer's protocol.
what could be the possible reasons?
I had success using the lab's protocol a few months ago (not consistently) but had difficulties getting the lab protocol to work again.
the manufacturer's protocol works and I find it is also more consistent.
thank you.
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