The time and temperature for PCR amplification are particularly concerning as we haven't been able to achieve the desired results. What should be done?
It depend on the primer sequence and you can design a primer of around 60°C Ta. The annealing time can be 30-45 sec. But the extension time should be more as the amplicon size is big.
I agree with Rajiv, your PCR annealing temperature depends on your primer length, its GC content and few more parameters . It is usually provided in an appendix supplied along with the primers, but practically you might not obtain a desired result at the same temperature. To resolve this issue we used to run a gradient with difference of 2 degrees e.g: 580C, 600C and so on. Further you can check for results at different cycles and MgSO4 Concentrations (generally 1.5-2 mM) , I believe you would be using Pfu Polymerase for amplification. You can provide a 1 min. annealing, 2 min. extension and 10 min. final extension. Vary these values accordingly. Cheers!!!
If you are going to try some annealing temperature for the first time and you do not have gradient cycler use below tool for calculation of annealing temperature, just input your Taq polymerase type and buffer along with primer concentration that you are using. Feed the primer sequence to this tool and you are near to the perfect annealing temperature for your PCR.
http://tmcalculator.neb.com/#!/
The optimal annealing temperature depends not only on primer sequence but also on choice of polymerase. A >6.5 kb amplicon most likely require something else than a standard Taq polymerase, such as Phusion Hot Start or Q5 high Fidelty Polymerase. Even with these highly processive polymerases you may need a 2-3 min extension step to get good amplification. The optimal annealing temperature for these two enzymes in is higher than for Taq. Also other long range polymerases such as LongAmp Hot Start Taq DNA Polymerase may provide a solution.
For calculating annealing temperature for your primers using different polymerases there are several online calculators such as:
http://tmcalculator.neb.com/#!/
Hope this helps. Let us know how things proceed for you!
Hi, amplifying something this big can be difficult. If you have some good quality template you can do a few simple tests in order to get the better conditions:
Try different temperatures (40 to 60, in intervals) and also a few different extension times (for your fragment 6 min should be adequate, but you can try less). If you have access to a PCR machine with gradient option you can do a lot with just one run.
But my real advice is to try to find an alternative to amplifying this fragment by PCR. I have seen a lot of people losing time and money trying to achieve this type of task. Depending on your budget, ordering your DNA from a company that can custom make it can be much more reliable and save you a LOT of time and money. You can even optimize codons or order mutated alternatives, depending on your project goals.
The DNA we are using is the human DNA and its approx. size is nearly 6 kb. We are using the Dream taq for PCR, but what if the target DNA is a human gene incorporated in a plasmid?
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