I could really use some feedback on the following:
My situation:
- I have 400 genomic DNA samples.
- They contain 60 highly-similar regions of interest (300 bp).
- I can amplify them all with just 3 PCR primer pairs: giving me a mix of
20 products (300bp) in each PCR reaction.
- I use different barcodes for all the PCR reactions and have extended my
oligo's with KEY and Primer A (forward) and B (reverse) sequences of 454.
- I do every PCR reaction twice, with different barcodes.
This gives me 2400 uniquely barcoded PCR-products of the same length (420bp). I would like to use one half/full titanium run on a 454.
I know that equimolar pooling is going to be tricky.
Now my question:
- Should I just mix all PCR products together, take a sample, do clean-up, sequence it and hope for the best?
- All PCRs that I have done will be quantitative, so I have end-point fluorescence for every sample: should I try and do equimolar pooling based on this value and then clean-up.
- or should I clean-up every sample separately, then use picogreen to measure concentration, then do equimolar pooling, and sequence?
Anyone have experience with >96 barcoded samples and how to do equimolar pooling, did you get extreme bias or was it doable?
Any feedback/suggestions and ideas are much appreciated!
My situation:
- I have 400 genomic DNA samples.
- They contain 60 highly-similar regions of interest (300 bp).
- I can amplify them all with just 3 PCR primer pairs: giving me a mix of
20 products (300bp) in each PCR reaction.
- I use different barcodes for all the PCR reactions and have extended my
oligo's with KEY and Primer A (forward) and B (reverse) sequences of 454.
- I do every PCR reaction twice, with different barcodes.
This gives me 2400 uniquely barcoded PCR-products of the same length (420bp). I would like to use one half/full titanium run on a 454.
I know that equimolar pooling is going to be tricky.
Now my question:
- Should I just mix all PCR products together, take a sample, do clean-up, sequence it and hope for the best?
- All PCRs that I have done will be quantitative, so I have end-point fluorescence for every sample: should I try and do equimolar pooling based on this value and then clean-up.
- or should I clean-up every sample separately, then use picogreen to measure concentration, then do equimolar pooling, and sequence?
Anyone have experience with >96 barcoded samples and how to do equimolar pooling, did you get extreme bias or was it doable?
Any feedback/suggestions and ideas are much appreciated!
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