PhiC31 integrase RNA prep
Jack Bateman
1. Linearize pETphic31-polyA with BamHI, precipitate, and resuspend in a small
volume of RNAse-free water.
I use about 1 microgram of linearized plasmid for each RNA prep, so I cut a lot in
advance. Note that this plasmid does not seem to prep very well - yields from a qiagen
midi column are consistently low in our hands.
typical digest:
90 l midiprepped vector
10 l reaction buffer NEB 2
2 l BamHI
- cut 3 hours, heat kill at 65C 15 minutes
- ethanol precipitate, resuspend in 10 microliters RNAse-free water
2. Transcribe RNA using Ambion mMESSAGE mMACHINE T7 kit.
typical reaction:
3 l linearized vector (1 g)
2 l 10 reaction buffer
10 l 2x NTP/CAP mix
2 l enzyme mix
3 l RNAse-free water
-incubate at 37C for 2 hours
3. Precipitate RNA.
-add 30 l RNAse-free water, 30 l LiCl precipitation solution from Ambion kit
-incubate in –20C freezer 30 min to 1 hour
-spin 15 min in cold centrifuge, wash 2x with 70% ethanol, resuspend in 10-20 l of your
favorite injection buffer
I typically get a little less than 20g of total RNA as measured by spec.
4. Mix RNA with donor plasmid.
I use a qiagen midi kit to prep donor vectors, then precipitate and resuspend in an
injection buffer. (I’ve tried preps from qiagen mini-spin columns and they degrade the
integrase RNA when mixed. You can, however, run these preps over a second
purification column, and that seems to make them useable). Final concentration of donor
vector when mixed with RNA is 400ng/l; we’ve used final RNA concentrations of
650ng and 950 ng in different experiments and gotten inserts. Check 0.5 - 1 l on a gel
before and after injecting to make sure the RNA was stable.