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Old 05-14-2015, 01:48 AM   #4
Al Merry
Junior Member
 
Location: Oxford

Join Date: Feb 2013
Posts: 3
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Hi Ruby, I decided against using the speedvac as I was wary about RNAse contamination. I was very happy using the Turbo DNAse. After tweaking the procedure, I ended up DNAsing using Turbo DNAse, then cleaning up with Qiagen minelute columns (using a few precautions to enhance purity), then precipitating in the presence of LPA as carrier. Recovery and quality were good, paper is submitted. Detailed method is:
DNAse up to 20ug in 100ul using 1ul DNAse according to Ambion Turbo DNAse instructions. Don't add stop solution after 37 degree incubation for 20 minutes, but proceed directly to next step (Qiagen Minelute cleanup). DON'T put on ice! (many forum posts cautioning against this)
Add 3.5 vols warmed RLT (@37 degrees; 175ul to 50 ul, 350ul to 100ul)
Add 1.5 vols EtOH (350ul if 50ul+175ul, 700ul if 100ul +350ul)
Add to minelute column
Spin 15s top speed
Wash 500ul RPE thoroughly (2mins, or roll - I found lots of forum posts stressing the importance of washing the rim of the column, to wash away all the salt which could be carried through as impurity later on). Spin
Wash 500ul 80% EtOH thoroughly. Spin
Spin, lid off, 5 mins, top speed
Elute with 14ul H2O. RT 5-10', spin 1' top speed.
I then needed to precipitate to concentrate my RNA:
1ul Linear Polyacrylamide (Sigma)
10ul NH4OAc
75ul H2O (can prepare these three as a mix and add 86ul)
250ul EtOH
-80 degrees 15 mins or -20, 3 hours
spin 4 degrees, 10'
2x wash 70% EtOH, RT 5', Spin 4 degrees, 5'.
Dry 55 degrees 5'
Resuspend in a suitable volume (5 or 10ul for me).

Hope that's interesting, good luck!

Al
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