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I followed the standard SYBR Green Protocol for doing a qPCR. For which I used

  • 10 uL of 1X SYBR Green Master Mix
  • Forward Primer and Reverse Primer (each at a final conc. = 8.5 uM)
  • Template (unknown concentration from an aptamer selection)
  • Made to 20 uL with water.

Performed the PCR amplification using

  1. 95 C Initial denaturation
  2. 95, 68, 72 - cycled 35 times.

But, one set of sample from my aptamer selection (probably high GC content) did not amplify at all. I checked on an agarose gel too.

Important Notes:

  1. My primer conc. is intentionally high - I have always amplified with such concentrations and it works perfectly using Vent Polymerase.

  2. My annealing temperature is also high at 68 C because my aptamer sequences tend to be GC Rich.

  3. Using Vent polymerase my amplification is very strong

On reading notes from vendors, I found out that the polymerase in the mastermix to be some variant of Taq Polymerase[1][3] (varies from vendor to vendor) and also it is known to poorly amplify GC Rich Sequences[2].

  1. https://www.thermofisher.com/order/catalog/product/4309155

  2. https://www.thermofisher.com/order/catalog/product/N8080241

  3. https://www.fishersci.co.uk/shop/products/powerup-sybr-green-master-mix/15366158

Hence, I wanted a suggestion from anyone working with aptamers, or if you have experience working with GC rich sequences on how I could amplify my sequences.

Much appreciated if I could have an answer before next week.

Thanks a lot for your help!!!

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  • $\begingroup$ Can anyone help with this if you work with GC rich sequences? $\endgroup$ – Somdeb Dec 19 '18 at 5:49

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