Genomic DNA Southern blot hybridization Alkaline Transfer Problem
|
Genomic DNA Southern blot hybridization Alkaline Transfer Problem
I am performing downward alkaline transfer for genomic DNA blot
hybridizations (and the transfers are great!), however, the hybridization
signal is just not there. I was wondering whether or not the alkaline
conditions (pH ~10-11) after the transfer (even after 2X SSC
neutralization) inhibits DNA/DNA probe hybridizations? No matter how much
2X SSC I use to neutralize the blot after transfer, the pH is still above
10. By the way, I am using the Boehringer Mannheim DIG kit, if that is of
any significance to alkaline conditions...
Original post
From what I understand the DIG-to-base link is alkaline labile
to allow you to easily and completely strip your blots. I don't
have my DIG manual handy but IIRC it requires dilute NaOH to
cleave the link in a quantitative manner. However, pH >10 is
definitely not going to do your DIG probe any good. I assume
you've checked the pH of your SSC and hybridisation buffer.
I'd suggest several soaks in x10 SSC after the transfer
until the pH of the "supernatant" approaches that of your SSC.
What buffer are you using for hybridisation? If its eg.
Church solution then make sure the phosphate achieved the
correct pH.
Do you really have to do strongly alkaline transfer?
When the instructions say neutralise with SSC they mean
*neutralise* with SSC (not just dip it in it and assume that
the pH has changed).
Good luck,
Bernard
Original post
SSC is very poorly-buffered; why not use SSPE instead? Alternatively,
use a pH 7.5 buffered Tris solution; perhaps like the one which you
used to use for gel neutralization before you started doing alkaline
transfers!
David J. Meyer, Ph.D
Original post
I gree, why not save a lot of tears and use tris? I do this evertime, 100 mm
Tris pH 7.5, and skip the SSC. One other point, you are using a charged
membrane aren't you? DNA will not stay on an uncharged membrane after
alkaline transfer, not at all.
All the best,
Original post
I am in an R&D lab at a biotech company where we have been researching
the best possible ways to perform non-rad Southerns and Northerns. We
utilize a biotin-streptavidin system with chemiluminescent detection. We
recommend upward alkaline transfers, though I am sure downward ones
would work too. The complete protocol for the Southern and detection
are at www.kpl.com in the 54-30-03 manual.
Good luck,
J. Ray
Original post
Last update 19-Jan-2002, Rating Good of 0 votes.
|
Write your comment
|
I have tried several DIG method adaptations and found that the Engler-Blum (1993)modified wash buffer and blocking buffer (0.5% roche blocker) in the immunological stage works nicely (3M NaCl, 0.1M maleic acid pH > 8). This may be party because the blocker is an acidic casein hydrosylate which acidifies the blocking solution, so although you pH it to 7.5 when you add in the blocker the pH falls below optimal. Another adaptation is to dilute the CSPD 1:500 and 'batch' the memranes thru a 5 or 10mL bath of CSPD in detection buffer. This seems to massively reduce background created when you drop CSPD onto the nylon membrane. I only use roche's positively charged nylons, and do a standard 20*SSC transfer overnight with a capillary blot - it works so why not?
Rating: Good
Reply
|
|
|
I have tried several DIG method adaptations and found that the Engler-Blum (1993)modified wash buffer and blocking buffer (0.5% roche blocker) in the immunological stage works nicely (3M NaCl, 0.1M maleic acid pH > 8). This may be party because the blocker is an acidic casein hydrosylate which acidifies the blocking solution, so although you pH it to 7.5 when you add in the blocker the pH falls below optimal. Another adaptation is to dilute the CSPD 1:500 and 'batch' the memranes thru a 5 or 10mL bath of CSPD in detection buffer. This seems to massively reduce background created when you drop CSPD onto the nylon membrane. I only use roche's positively charged nylons, and do a standard 20*SSC transfer overnight with a capillary blot - it works so why not?
Rating: Good
Reply
|
|
Related resource
Southern blot background
DNA southern blot hybridization
Plasmid vector unspecific hybridization
Protein blocking solution for DNA hybridization
Hybridization buffer recipe
Digoxigenin DIG Southern Blots
Gene copy number by slot-blot hybridization
DNA hybridization using 100 bp insert as probe
|
