Pippin Prep - cDNA musings...

[DrDTonge]

Dear all,

We've purchased a Pippin Prep and are awaiting training to use the 3% for size selecting our small RNA libraries. Briefly, these comprise of 18 - 38nt inserts with adapters making a total of 60 -80nt following reverse transcription.

We're informed that we can use the Pippin to size select straight from the cDNA reaction although I have a query about this...

Our usual method is to run the cDNA down a denaturing gel (TBE-UREA) and excise the cDNA fragments in line with a denatured DNA ladder. Therefore both reference and sample are denatured.

Unless I'm missing something obvious, I can't work out how the native gels in the Pippin Prep can achieve this against a native DNA ladder...the first strand cDNA would be ss, whilst the reference ladder would be ds.

What am I missing???

D :-)

[mehdik]

ssDNA selection on Pippin

The way you can use the Pippin for selecting ssDNA molecules is to empirically determine at what size they come out compared to the dsDNA ladder. It works well if your process calls for the same size selection range over and over. If it varies, then there might be a lot of optimization.