DNA isolation/purification troubleshooting.
Posted 20 May 2012 - 01:14 PM
Btw, what happens when you use a slightly high (~75%) or slightly low (65%) EtOH for washing?
Posted 20 May 2012 - 01:56 PM
Posted 21 May 2012 - 12:55 PM
You can also do column extractions (from a kit) of DNA that will give you clean DNA, but they are relatively expensive.
Also, if you have too much EDTA in your samples, this will inhibit the PCR by chelating the Mg ions needed as co-factors for the DNA polymerase. Try diluting your samples 1:100 or 1:1000 to see if that works.
Posted 21 May 2012 - 05:24 PM
I will try diluting the samples first and see if that works. If not then I'll do a couple of phenol extractions.
Also, will using a lower percentage of ethanol (65%) for the wash steps affect the DNA purification process?
Posted 23 May 2012 - 12:51 PM
The percentage ethanol is designed to keep the DNA precipitated but still dissolve salts. I don't think 65% instead of 70% would have too much effect, but I can't be sure on that.