I'm using Nextera XT on environmental samples for metagenome sequencing, with only a few samples (2-5) per run. We decided to skip the bead normalisation step in our XT runs and normalise the pooled library manually, following the Nextera DNA protocol. However, the PCR product (CAN) of some samples is at a lower concentration than the 2nM suggested for pooling and denaturing. My current plan is this:
My two main questions are:
Thanks,
Dylan
- Pool and normalise the libraries by calculating the required volume of each eg
- 30ul of 0.66nM product
- 20ul of 1nM product
- 10ul of 2nM product
- Calculate the concentration of the pooled library (in the above example 1nM) and use the equivalent calculated volume for denaturing (ie 20ul of 1nM instead of 10ul of 2nM)
- Add the required volume of HT buffer to result in 1000ul at 20pM
My two main questions are:
- What concentration of NaOH should I use? Should I just use the equivalent volume of 0.2 N so that the final concentration is 0.1 N, or should I lower the concentration to match the lower DNA concentration?
- Should I rather increase the PCR cycles to try and generate more product?
Thanks,
Dylan
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