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Sensitive, Consistent and Reproducible Method(s) for Extraction of gDNA from Low

Blood gDNA Extraction Consistency Low Volume

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2 replies to this topic

#1 Fluoresce

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Posted 27 April 2013 - 06:35 PM

Hello all,
Have tried to extract gDNA (frozen whole blood) (less than 300 ul). I have had not much luck to have a sensitive, consitent/reproducible extraction with methods such as salting out or magnetic beads. It is critical to use low volumes for my purposes. Can anyone suggest another method such as 96 well format extraction that has been tested and showed good results as far as reprodicibility/consistency? I'd really apprecite your inputs.
I can also use plasma (super low volume).

Thanks,
FL

#2 AquaPlasmid

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Posted 31 May 2013 - 08:16 AM

AquaPreserve will work. Here's the protocol for DNA extraction from frozen whole blood.

1. Lyse the blood cells

Add 0.25 ml of AquaPreserve to 0.25 ml of fresh or frozen whole blood in a 1.5-ml microfuge tube. Vortex or shake to mix well or until the frozen blood sample is completely thawed in AquaPreserve (Do not thaw the frozen blood without mixing with AquaPreserve or the RNA will be degraded during blood thawing. However, if you are only interested in blood DNA extraction, the blood sample may be thawed, aliquoted and then mixed with AquaPreserve.).

2. Remove the proteins

Add 0.125 ml of ProSink (#9030) to the AquaPreserve lysed blood. Vortex for 1 min to mix well and incubate at 22 °C for >30 min (Blood DNA is now stable at 4-22 °C for a year, and blood RNA is now stable at -80 °C forever, 4 °C for 2 weeks, 22 °C for 7 days.). Centrifuge at 14,000xg for 10 min to pellet the proteins (save the pellet for protein recovery with ProMelt (#1115), if desired).

3. Pellet the DNA/RNA

Pour the supernatant (~0.7 ml) to a 1.5-ml microfuge tube preloaded with 0.8 vol (~0.56 ml) of isopropanol and vortex to mix. Centrifuge at 14,000 xg for 10 min to pellet the DNA/RNA. Decant to discard the supernatant (or save it for small molecule analysis) and rinse the DNA/RNA pellet with 70% ethanol twice. Suspend the DNA/RNA pellet in 50 ul of TE buffer or deionized water.


#3 Fluoresce

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Posted 01 June 2013 - 07:26 PM

Thanks a lot for your precise and detailed reply on my queston regarding extraction from low volume blood. Your protocol seems very promising. Thanks again!
FL





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