Dear colleagues,
I'm trying to extract genomic DNA from amphibian muscle tissue without getting any RNA contamination. I'm using two different extraction methods:
1) Qiagen DNA Tissue Kit
2) Phenol chloroform
Someone in my lab told me to add 1uL of RNase A (10mg/ml) at the end of the extraction protocol and incubate 10 min at RT (regardless of my final elution volume). By using NanoDrop I mesured DNA concentration of my samples before / after adding RNase A, and absorbance curve gets really bad after.
For this reason, I'm not sure if that's the appropiate way of using RNAse A.
Does someone have any recommendation?
Thank you very much!
RNase A treatment is the most efficient way to remove the RNA from the DNA preps. Since the free dNTP also have UV absorption and cause hyperchromic shift, it is not surprising that you has a bad curve after the RNase A treatment. Your can use ethanol precipitation to remove the free dNTP and even better do a step of phenol chloroform extraction to remove the proteins (e.g these existed in tRNA) and followed by ethanol precipitation.
Hope this helpful.
http://oxfordgenetics.com/cloning-resources/cloning-guides/rnase-treatment-rna-removal
Hey Santi!
We add 2ul or RNAse A to a volume of 650ul (extraction buffer), and incubate 10min at 37ºC.
Better check on a gel, runing untreated sample besides your sample treated with RNAse. You will see then if there's still some RNA in your sample, and also the integrity of your gDNA.
Hope it works. Good luck!
Thank you very much!
@ Helena: So you add RNase A right after digestion with Proteinase K and then proceed as usual with your gDNA extraction protocol, right? Which is the concentration of your RNase A (mg/ml)? Which company do you purchase it from? Thanks a lot! :-)
RNase A never work during extraction process,,it must be used after isolation,,IN MY OPINION add 2 ul RNase A incubate for 1 to 2 hour at 37,then purify your DNA again..Purification methods vary,you can choose one feasible for you..best one is by mean of DNA beads that selectively bind DNA and all other materials removed.best luck
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