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Basically you don't need to do any more than ensure that the blood is fully frozen and then fully thawed. The quickest way to do this is use a dry ice/isopropanol bath (dry ice pellets in isopropanol, take care with tube labelling, this will remove most pen written labels) and a 37 degree bath, dunk the tubes into the baths alternately. You could also use liquid nitrogen, but it is a bit more dangerous, isopropanol at -80 is also effective, but warms up rapidly unless you work in the freezer.
#151968 Help: Best way to disrupt blood cells to generate blood debris and gDNA to be us
Posted
on 10 March 2013 - 12:12 PM
#151966 Help: Best way to disrupt blood cells to generate blood debris and gDNA to be us
Posted
on 10 March 2013 - 11:43 AM
Freeze/thaw should work well - the ice crystal formation is enough to lyse the cells fairly effectively. I would do about 3 cycles for complete lysis.
#150077 Qiagen column eluted DNA gives higher concetration than the original sample
Posted
on 10 February 2013 - 07:21 PM
I follow kit with a few exceptions. I allow equilibration buffer to sit in column longer, I spin down ethanol at maxi speed for 2x time suggested, I allow elution buffer to sit in column for ~5minutes, I then rerun same elution buffer back through column. Qiagen is a good kit, following the protocol should yield good results.
Bob has a good point. Make sure your dilution calculations are correct.
Good luck
Bob has a good point. Make sure your dilution calculations are correct.
Good luck
#150076 Qiagen column eluted DNA gives higher concetration than the original sample
Posted
on 10 February 2013 - 06:55 PM
I would expect a column DNA extraction (especially for pre-extracted and spiked DNA) to be about 30% of total - if you eluted in 1/10th the original volume and you have 30% yield - that will give you 3x the concentration, but only 1/10th the volume so they total yield is less...
