8 replies to this topic

[alex84xela]

Hello everyone !
I'm having biiiiiig trouble with my southern blotting.
First how i proceed :
- Digest genomic DNA (BamHI) and make a 0,8% agarose gel with 10µg of DNA per lane
- Transfer using a very direct solution : 2 times 0,25M HCl 10 minutes, 2 times 0,4M NaOH 10 minutes wash before a 0,4M NaOH transfer, followed by 5-10 minutes 2x SSC 0,1% SDS wash
- I'm using Genescript + membranes
- Once transferred, i make a 0,7 kb probe using Amersham kit (kleenow and random primers).
- Pre hybridation for at least 2 hours in 20 ml church (1% BSA, 200mM NaP, 15% Formamide, 1mM EDTA, 7% SDS)
- I throw away 10 ml church and put my probe in the left church
- 16 hours hybridation
- 2 times 10 minutes 2x SSC 0,1% SDS
- 2 times 10 minutes 0,1x SSC 0,1% SDS
- Exposing

I get a weird result :
I test my probe with the source of my probe : works fine
In lanes with DNA i get dark smears, and sometimes some very low resolved bands, hard to distinguish from the background noise
For the DNA ladder, i'm using lambda phage digested (nothing is detected : 20kb to 2 kb ladder) and a plasmid from our lab digested (making 1kb, 0,8 kb, ...). I get a very intense signal on the 1 kb fragment of the ladder ! But nothing on other sizes.

If it were simply background, i would have all ladder sizes making a signal.
It's really tricky...
Can somebody help out ?

i'm a poor desperate PHD student  

[hotstuffdb22]

Hello,

How long do you transfer for and what buffer do you use?  How large are the paper towels on the transfer apparatus?  Do you check the gel post transfer to make sure transfer is complete?

[alex84xela]

Hello and thanks for your reply.

I transfer overnight usually, but one try was a short 3 hour transfer too.
I'm using a large amount of paper towels, something like 10 cm high.
My transfer buffer is simply 0,4M NaOH (this protocol worked well a few years ago tells my boss, showing me nice results).
I didn't check the gel post transfer, but it's very thin.

Your guess is it would be a transfer problem ?

I think my main problem is a signal/noise problem, but this probe worked well already in other hands. Transfer seems ok :
There is a smear in each lane containing DNA, and nothing in empty lanes, and a high signal on the 0,8kb band of the ladder (not on the other bands) and on my positive control lane containing only a digested plasmid that was used to make the probe (signal at the right size too !).

Edited by alex84xela, 30 May 2010 - 11:00 PM.

[alex84xela]

Some people will send me alternate protocols to test (and other membranes)...
I also found this on a manual :
"In our experience, this step is sometimes unnecessary, but we recommend including it if there are problems with high background or weak signal. Although nylon membranes are not flammable and do not require a vacuum, you may wish to use a vacuum to trap any residual formaldehyde fumes.
1. Rinse the membrane in 2X SSC or 2X SSPE to remove residual agarose. Any agarose remaining on the membrane may cause a high background.
2. Place the membrane on a clean piece of filter paper to remove excess buffer.
3. Bake the membrane at 80°C for 1-2 hours. While it is not required, the membranes will dry faster under a vacuum. At this point the membranes may be stored for future use.
4. Wet in 2X SSC or 2X SSPE before proceeding to prehybridization."

I don't think there is agarose left on the membrane, and i will try baking it before prehyb and hyb...

[alex84xela]

So i went on trying to troubleshoot this problem : i changed BSA for fraction V BSA, i used a Hybond N+ membrane.
Didn't work.
Troubleshooting still ongoing

[mdfenko]

have you tried blocking with salmon (or herring) sperm dna?

[alex84xela]

Not yet, I was planning to do that, very good idea !
The only other person doing southern blots at my institute blocks her membranes.... without BSA. She uses NaP buffer with nothing else inside. No DNA, no Proteins. Weird. But hers do work without any background !!! It's so unfair !!!
She sent me her protocol i will try it too.
I will also wash my membrane for a longer time, maybe it'll help out...

[alex84xela]

Before salmon sperm dna i am trying a few things, i think i'm on a good way : i started washing at higher temperatures, 75°C !!!! Very long washes in 0,05x SSC allowed me to get a signal only on my positive control, my test lanes lost their signal though. I'll try again washing a bit more gently.

[alex84xela]

Hi everyone !

I succeeded and got a readable result despite a big smear in the background.
I had to block 24h in my church solution for that, and it worked only with Fraction V BSA and long exposure.
I still do not know what my problem was but I will go on with my project.

I tried salmon sperm DNA, it didn't change anything, still got a smear.