2 replies to this topic

[Pheebz Pagnol]

Hi there,

I would like to screen ES cells for my knock-in mutation (or rather for the presence or absence of the preceding Neo cassette) by Southern. The digest of the genomic DNA looks fine. I use Hybond-XL as membrane and use 10X SSC for transfer. I prehybridize with church buffer (0.5M NaPi, 0.05% SDS, 0.5 mM EDTA, 0.1 mg/l salmon sperm DNA, 0.01% BSA) over night (to reduce the background) at 65°C, hybridize o/n at 65°C, wash with Church wash (0.04 mM NaPi, 1% SDS).

So far the procedure worked more or less (for other probes, other digests). But now with the new ES cells I get a completely clean membrane except for a signle marker band   Since I see the marker, I would assume the transfer worked, right?

I cannot pre-hybridize too long, can I?
Would it make a big difference, if I washed with 0.4% SSC, 1% SDS (but that would be even more stringent, wouldn't it)?

Well, these are basically all the leads I got as to what is going on. I am sorry if I am asking stupid questions (e.g. strigency) but I am very tired and very worried. My boss will kill me because the probe used to work (not in my hands, though) and Southerns are sooooooooo easy (compared to Westerns, he says).

I'd appreciate any help.

Best wishes,

Pheebz

[Rsm]

Hi,
I've had similar problems after I made new Church Buffer. I realized that 0.5M Na2HPO4 means in fact 0.5N sodium, that is 0.25M Na2HPO4. So I added too much salt...  See also here. But there seems to be many different protocols for Church buffer.
I don't think that O/N prehyb is a problem.
Cheers,
Minna.

[bochum]

Even i had this problem, and the frequency is more when i prepare new buffer. so check which stuff you changed from your previous set ES clone and the set. it may be buffer,temp,time of incubation,

Preparation of DNA also very important step,if the DNA is not dissolved properly this also leads to bad blot mainly because of bad digestion of DNA.

Happy blotting