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|01-05-2017, 09:16 PM||#1|
Join Date: Jan 2017
Increase yield in library preparation
We are using the nextera XT DNA library prep kit in order to produce sequencing libraries from RNAseq experiments. For the purpose of our study, we need around 10ug of library products. I know it is quite a lot for sequencing libraries but more steps will be performed before the sequencing and thus we need this amount.
We first thought performing more PCR cycles in the Reduced-Cycle PCR Amplification of the nextera XT kit. But it failed and the size of the library was shifted.
We then decided to purify these products and performed a second “ classic” PCR.
However, whatever our conditions are, the PCR seems to saturate at 200ng of PCR products (around 9ng/ul).
We already tried several things:
- change the PCR enzyme (Kappa enzyme, Ex taq)
- change the primers
- change the purification step after the amplified PCR (AmpureXP beads, PCR clean up kit)
- change the volume of PCR reaction (50ul or 100ul) to increase enzyme, dNTP or primers quantity
- change the amount of starting material (10ng, 50ng and 100ng).
All of the tests saturated around 200-250ng.
We have also checked with an agarose gel performed before the purification step that we are not producing a lot of primers artefacts that might saturate the reaction. But no, there are not so many primers-dimers.
Does any one have an idea why the PCR saturates at 200ng ?
Thank you for your help
|01-06-2017, 03:58 AM||#2|
Location: Washington DC
Join Date: Oct 2009
There is no inherent limit on how much PCR product you can generate. As for any chemical reaction, the amount of product is dependent upon the amounts and concentrations of the reactants (template, primers, dNTPs, enzyme) and reaction conditions (buffer, time, temperature). Without knowing your reaction mix it's difficult to say which component is limiting, but my money is on the primer or dNTP concentration. I'd try increasing the amounts of those reagents.