29 April 2014


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ddRADseq debugging

Pooling and size selection

  • Pooled all remaining ligated sample without AMPure bead purification
  • Ran on 1.5% agarose gel for 3 hours
  • still no DNA visible in sample lane when I visualize gel
Gel image
Gel image

  • Excised gel fragments for 275, 375 and 475 bp +- 25bp
  • QIAquick gel purification

DNA concentration from Qubit

Sample Stock conc (ng/ul) Total DNA (ng)
P 8.78 351.2
P-200 0.055 0.5
P-300 0.066 0.6
P-400 0 0

Total DNA is in 40 ul for sample P and 10 ul for P-200, P-300 and P-400

Key points:

  • DNA is lost during AMPure purification step during pooling
  • From Bioanalyzer, expect about 10% of DNA to be in the 200 bp window, so could maximally recover about 8 ng, yet recover less than 1. So, even the gel purification is problematic.

Seems that I need more input DNA…though I’m using within recommended range (100 - 500 ng) from ddRADseq protocol. What gives??




Phone call with SK - discussed analyses to finish Silene ms

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