4 replies to this topic

[antaine76]

Hi everyone,
i'm looking for a good southern blot protocol with all the secrets of this technique. I never did it but I know is not too difficult.
I'd like a protocol that show me the "way to do" fro digestion to exposure.
Consider that I have to do this Southern on human genomic DNA looking for a band really AT rich (over 70%). I have this sequence cloned in a vector (identified to be a mRNA) and I want to find it on the genome.
Tell me how to design the probe too.
Thanks in advance

Antaine76

[leahf]

Hi Antaine
what sort of label are you planning to use? if you are using the DIG (non-radioactive) label, try Roche's 'DIG application manual for filter hybridization' - it gives pretty thorough overview of all the different ways to make your probes, and complete protocols for hybridization and detection as well.  I guess the same protocols would apply for radioactive labelling, except you may need to modify the DNA and probe concentrations.
you can get the manual through the company's represantatives or online - go to http://www.roche-app...e.com/index.jsp , pick molecular biology -> DIG reagents and kits-> literature.
and a tip - you do NOT have to purchase all their buffer and wash kits - they are very expensive and you can easily make them yourselve.
luck
leah
P.S. - i am not connected with Roche  in any way...i've been using this manual myself and found it very helpful.

[rocky]

Hi everybody,

I am doing a Southern blot using Qbiogen positive membrane. I made a mistake while putting the transfered membranes in the baker during 7 hs (instead of maxi 2h). I don't know if it was damage ? And what may be the problems to these membranes ?

Thanks in advance for your answer !

[pcrman]

Just go ahead and do a hybridization to see what you will get. Most likely you will still get good results.

[rocky]

Hi everybody,
I did the hybridation, those membranes were bad in comparison to others. So, be careful and respect the protocol !