I am getting zero DNA yield after using qiagen purification columns. I finally traced the problem to NEBuffer 3.1, but pH doesn't seem to the cause.
Essentially, I observe:
3 ug of DNA in 50 uL of water ->
qiagen purification column ->
1.5-2.3 ug of DNA
In comparison:
3 ug of DNA, 5 uL of 10X NEBuffer 3.1, bring to 50 uL with water ->
qiagen purification column ->
zero DNA
I thought it was a pH problem -- high pH can cause low efficiency. But I don't think pH is the problem. Because pH strips and qiagen's pH indicator say my pH is okay (pH
Good morning! For DNA precipitation, the pH buffer should be high, around 8. Have you tried using sodium acetate precipitation for DNA extraction and digestion?
Hi, Ruslan Kalendar The Qiagen column purification kit begins with diluting the DNA sample in 'PB buffer' which contains guanidine hydrochloride and isopropanol. However, my DNA sample still does not behind the column despite this step.
Hi, Zahra Jalali Javaran For DNA to bind a silica column, the pH must be low, not high. I had not considered precipitation. I may try that. I was planning to try a different kit next.
I gave up on the column kits and did NaAc+Isopropanol+Ethanol precipitation and it worked well, although it takes much longer than the column kits:
2.5 ug DNA in 50 uL water -> alcohol precipitation -> 80% recovery
2.5 ug DNA in 50 uL water with 1X NEBuffer 3.1 -> alcohol precipitation -> 80% recovery
Ruslan Kalendar
It's not so much an efficiency issue, it's that I recover 0% of my DNA when I add digestion buffer to my DNA sample. But I recover 50-70% of my dna with identical setup except no digestion buffer. I still don't understand why digestion buffer causes no DNA binding.
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