DDRT-PCR Northerns

Michael Gerdes mg690769 at bcm.tmc.edu
Mon Jun 26 14:59:04 EST 1995


In article <3sil5t$gsq at bubba.NMSU.Edu> smori at nmsu.edu (Shahram Mori) writes:
>Dear Molbionetters and sufferers of DDRT,however the northerns don't work. How many people have 
>observed this out there? What did you do? Strauss et al in PCR Methods 
>and Applications said that they ahd given up doing Northerns without 
>giving a clear reason why. 

Shahram- here's what I have done and have had very good sucess-
it may seem like unnecessary work, but spinning your wheels with 
failed expts. is worse.

take your purified band and re-PCR
TA-clone 1ul into pCRII (invitrogen).
resrtiction cut a miniprep to insure insert size/presence.
do a maxi prep to get a fair amount of working material.
use maxiprep DNA for restriction digest, cut up >10ug.
isolate insert
random prime with 32P-dCTP
use this for your northerns.


reagents used include columns from qiagen, random prime hexamers from 
Boeringer Mannheim, gel/nucleotide clean up spin tubes from qiagen, and
the cloning vector from invitrogen.

i have heard of a lot of people having problems with their northerns, and 
i think a lot of it stems from wanting to take the fast apporoach to 
getting a labeled probe, and it fails frequently.  also, by going to the 
trouble of subcloning the pcr product, you are now in position to a> 
generate riboprobes for in situ's, b>end label the insert for screening a 
library, c> sequence the insert to see if it can be identified.

I have had very good sucess by this method- of 17 isolated bands, 8 have 
been confirmed by northerns.  Only 2 were undetectable by northerns.  
aslo, i just isolated clones from a library, with relative ease.  
finally, i was able to sequence all of them in a matter of a month, so 
now know that none of them are the same thing.

hope this helps,
will gladly provide further assisstance through e-mail if needed.

good luck

MiJoGe
mg690769 at mbcr.bcm.tmc.edu
Dept. Cell Biology
Baylor College of Medicine





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