ERIC HANSON re:RNase protections

Jillian Johnston johnstoj at acs2.acs.ucalgary.ca
Mon Jun 26 00:02:16 EST 1995


Hi Eric, I tried e-mailing but the message was returned 'Host
Unknown".
In answer to your question -
1- We purify the labelled probe with RNAid from HB101 (similar to
Geneclean).  We recover the target RNA from Xenopus oocytes and
ferts using a standard LiCl procedure.
2- We use 50uCi of 35S-CTP to transcribe linearized DNA in a 20ul
 rxn, again using standard probe synthesis methods.  We don't 
add cold CTP to the rxn.
3- Our target is very abundant since we inject it at
5ng/individual.  The assay will protect in the order of 0.5ng of
our target but this must be determined for each batch of probe.
We just do a rough mathematical determination but some folk
titrate their target RNA to find out how much RNA saturates the
probe.

The procedure we use - 
Probe at 100000 cpm/ul
Dry down the target RNA
Resuspend in 20ul hybridization mix (19ul hybridization buffer +
1ul probe)
Hybridization buffer
8.3ml formamide
0.8ml 5M NaCl
0.8ml 0.5M PIPES pH6.4
0.1ml 0.1M EDTA pH8

Incubate overnight at the right temp for hybridization of your
probe. (50C is usually about right).
The next morning, add 0.35ml digestion solution -
10mM Tris pH7.6
5mM EDTA
0.3 NaCl
40uG/ml RNaseA
10U/ml RNaseT1 (this enzyme may or may not be necessary.  It's
expensive so you should check to see if it will cut often enough
to be worth it)

Incubate 30 min @ room temp (or 28C)
Add sequentially 10ul 10% SDS and 5ul 10mg/ml proteinaseK.
Incubate as above for 10 min.
Phenol/chloroform once.
Add 4ug yeast or E.coli tRNA and 1ml cold EtOH..  Chill at -70c
for >10min.
Spin, wash with 70% EtOH, spin and air dry.
Resuspend the protected RNA in 4ul Stop Solution (95% formamde,
0.10% bromophenol blue, 0.01% xylene cyanol).
Heat the sample to 95C immediately before loading onto a 5%
urea/acryamide gel.  You can use either shark's tooth combs or
square wells.

Please feel free to ask for help whenener you need.
Jill



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