What kind of efficiency should i expect from NaAc pptn of DNA?
measuring crude chelex extractions of insect legs i get Nanodrop conc of around 30-150ng/ul, but really low 260/280 ratios (about 0.9).
Following the extraction with a ppt/EtOH wash, the ratios improve ( around 2), but concentration plummets to 5-10ng/ul...
is this normal?
Protocol is:
grind leg in 20ul Chelex/Prot K
heat 55deg/15min, 99deg/10 min
10min in ice
spin 5min 18000g, 10ul supernatant = 30-150ng/ul DNA
Add NaAc and EtOH, final volume 200ul
incubate 10min -20deg
spin 30min 18000g
remove supernatant (no pellet visible), wash with 50ul 70% EtOH
spin 15min 18000g
remove supernatant, dry 10min 37 deg( until no moisture visible in tube)
resuspend in 10ul sterile water (5-10ng/ul).
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