We are trying to extract genomic DNA from T4 but the end product after attempting resuspension in milli-water is a cloudy 'bundle' of DNA that will not dissolve into solution.
General process: High titer sucrose-purified T4 stock is diluted using a Tris, Mg, NaCl buffer (7.2) and extracted using equal volumes of phenol:chloroform 3 times. Gently rocking by hand for ~3 minutes followed by centrifugation in a benchtop centrifuge at 13.3k rpm for 5 minutes to form protein layer. After phenol extractions, the volume is back extracted with chloroform to remove contaminating phenol. Glycogen, sodium acetate (0.3 M final), and Ethanol (70% final) are used to precipitate the DNA overnight at -20. DNA always precipitates well and there is a good sized pellet after centrifugation in the morning. We perform two washes with cold 70% EtOH and allow to air dry for 20m at RT before attempting resuspension in H20 for 1 hour.
However, after one hour (or even overnight), the DNA remains in a semi-translucent, milky 'bundle' in the H20 and nothing we've attempted will bring the DNA further into solution. We've tried proteanase treaments, heat, resuspending in same TMS buffer.
When dry, high molecular weight DNA pellets are hard to redissolve. I'd try shortening the air drying (even if it means leaving some residual ethanol in), leaving out the glycogen (not needed if dealing with relatively large concentrations of DNA) and the Mg.
Hi Alejandro, thank you for your helpful reply. I will try your suggestions! Can you help me understand what about the leaving out the Mg might aid resuspension?
Oh sorry, no theoretical grounds for it or anything. It's just that years ago I once had a similar problem resuspending phage lambda DNA that had been precipitated from crude preparations. I no longer remember how the issue was solved, but I do recall the presence of Mg++ as one of the factors we looked at.
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