Bizarre RNAse cleanup problem
Hello everyone,
I am prepping some samples of plant genomic DNA for illumina sequencing. My extracts (CTAB) are fine with plenty of high molecular wheight DNA however, EVERY time I go to do an RNAse cleanup of my samples my DNA I get an insoluble pellet, and my DNA somehow loose my DNA. I am pretty confident that I dont have DNAse contamination in my RNAse.
Thinking perhaps my loss could stem from my the drying steps etc of a separate RNAse clanup, I just recently added the RNAse to the aqueous phase of my extract (CTAB) prior to the isopropanol step. Then finished with the 70%ethanol wash and resuspension.
When I added as little as 1ul Qiagen RNAse A to 4ml of aqueous phase (1hr at 37degrees then 15min at 70degrees), the end result was a large insoluble pellet. Adding 4ul of RNAse A produced an even larger insoluble pellet. The same exact extract (I had split each CTAB aqueous phase into 2 tubes) without RNAse treatment produced a clear easily dissolved pellet.
What is it in the RNAse A that is making this nightmare of JUNK in what should be a clean DNA pellet? I think my problems with recovery are tied to this gunks appearance. I am working with very limited moss tissue and am fighting for every microgram of DNA I can get.
Previously people in my lab have used the RNAse cleanup protocol successfully dozens of times, so its either something to do with my samples, or Im bungling it somehow...I would appreciate any advice!
I am prepping some samples of plant genomic DNA for illumina sequencing. My extracts (CTAB) are fine with plenty of high molecular wheight DNA however, EVERY time I go to do an RNAse cleanup of my samples my DNA I get an insoluble pellet, and my DNA somehow loose my DNA. I am pretty confident that I dont have DNAse contamination in my RNAse.
Thinking perhaps my loss could stem from my the drying steps etc of a separate RNAse clanup, I just recently added the RNAse to the aqueous phase of my extract (CTAB) prior to the isopropanol step. Then finished with the 70%ethanol wash and resuspension.
When I added as little as 1ul Qiagen RNAse A to 4ml of aqueous phase (1hr at 37degrees then 15min at 70degrees), the end result was a large insoluble pellet. Adding 4ul of RNAse A produced an even larger insoluble pellet. The same exact extract (I had split each CTAB aqueous phase into 2 tubes) without RNAse treatment produced a clear easily dissolved pellet.
What is it in the RNAse A that is making this nightmare of JUNK in what should be a clean DNA pellet? I think my problems with recovery are tied to this gunks appearance. I am working with very limited moss tissue and am fighting for every microgram of DNA I can get.
Previously people in my lab have used the RNAse cleanup protocol successfully dozens of times, so its either something to do with my samples, or Im bungling it somehow...I would appreciate any advice!
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