Recently I have been isolating genomic DNA from human breast tissue samples via phenol/chloroform extraction. After washing in ethanol, a large white pellet is clearly visible and is resuspended in TE Buffer. However, when I quantitate the DNA I get a very low yeild (e.g. less than 75ng/ul) and a 260/280 ratio of anywhere from >2.17 to 3.75. Any idea on (1) why the low yield from such large pellets, and (2) how to acquire purer DNA?
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#2
Curtis
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Posted 07 June 2012 - 07:58 PM
1- how much TE do you add? if you add a lot then it will affect the concentration
2- is your phenol new? is it yellowish or clear?
3- do you homogenize the tissue?
4- 75ng/ul is still not bad, and your purity is also OK. what is your spectrophotometer?
2- is your phenol new? is it yellowish or clear?
3- do you homogenize the tissue?
4- 75ng/ul is still not bad, and your purity is also OK. what is your spectrophotometer?
#3
Pkerns
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Posted 11 June 2012 - 12:48 PM
If you are drying your DNA sample aggressively (heat block) or for a long period after the DNA precipitation via the ethanol precipatation following phenol/chloroform reaction it's possible that your DNA is just taking a while to go back into solution. You might try adding the TE and letting it sit for a while at room temp, agitating the tube, then go read the sample.
Edited by Pkerns, 11 June 2012 - 12:48 PM.
