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rudzik79 · 08-16-2017, 03:33 AM

Thank you. For a standard I used microbial DNA that has worked before and was 16s sequenced. I checked everything for DNA intergrity first, it's fine.
The polymerase is not the same, but it has proofreading abilities and was used by me to prepare other libraries (iProof bioRad) or was recommended online as a substitute for KAPA (NEBNext).

My GRADIENT TOUCH-DOWN PCR was completely empty, and I used 15 cycles for touch down (68-61 in one corner and 58 down to 51 i the other with 5 intermediate touch-downs in between) and 20 regular cycles in the lowest temperature. That's how I normally do optimisation for tough products. This time it was empty.

Now my friend is bringing me primers that are known to work, because I have completely no idea what to do next... KAPA is not readily available in my country now. Plus, it's twice the price of NEB. And I have plenty of iProof that is nearing expiry date so I could use a lot of that too.

08-16-2017, 03:33 AM	#3
rudzik79 Member Location: Poland Join Date: May 2016 Posts: 10	Thank you. For a standard I used microbial DNA that has worked before and was 16s sequenced. I checked everything for DNA intergrity first, it's fine. The polymerase is not the same, but it has proofreading abilities and was used by me to prepare other libraries (iProof bioRad) or was recommended online as a substitute for KAPA (NEBNext). My GRADIENT TOUCH-DOWN PCR was completely empty, and I used 15 cycles for touch down (68-61 in one corner and 58 down to 51 i the other with 5 intermediate touch-downs in between) and 20 regular cycles in the lowest temperature. That's how I normally do optimisation for tough products. This time it was empty. Now my friend is bringing me primers that are known to work, because I have completely no idea what to do next... KAPA is not readily available in my country now. Plus, it's twice the price of NEB. And I have plenty of iProof that is nearing expiry date so I could use a lot of that too.