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