New to the forums, just looking for some help in an area where I have been struggling.
My lab is trying to use Southern blotting for genotyping confirmation. At the point where I joined the lab and took over the blotting, we had already chosen and ordered primers for probes, and I cobbled together a protocol from my PI's protocols, the internet, and instructions that came with our solutions/membrane.
I am getting successful labeling of the Lambda HindIII ladder and of amplified PCR fragments, so I know that the problem isn't to do with labeling the probe or DNA transfer or gel runs or anything. What we're seeing is bright labeling on the ladder and some smearing and background in the sample lanes but no distinct bands anywhere.
Quick outline of protocol:
- Genomic DNA from whole tissue by phenol/chloroform extraction, ethanol precipitation, and resuspend in TE. Check on a Nanodrop for concentration - DNA is clean and pure.
- Digest DNA with Sac I up to 24 hours - have tried several different amounts, anywhere from 10 mg to 40 mg, changing volume accordingly.
- Run on 0.66% agarose gel at 50V for 8 hours; imaging shows clean digestion, no excess of undigested DNA.
- Denature in NaOH/NaCl solution for 1 hr
- Neutralize for 30 minutes
- Blot onto Hybond - N+ membrane overnight via neutral transfer.
- Crosslink blot, rinse, and let dry before storing.
- For probing, re-wet blot in 2x SSC.
- Lay blot on a bit of nylon mesh and place in a hybridization bottle.
- Pre-hyb with 7.5 mL Amersham Rapid-Hyb buffer for ~30 minutes (have tried this up to overnight with no difference on outcome) at 65 degrees C.
- Mix thawed probe (~15 ng) 50/50 with formamide and denature at 95 degrees C for 5 minutes, quick cool on ice, and pipette into hybridization bottle.
- Hybridize overnight at 65 degrees C.
- Rinse 3x 10 minutes in 2x SSC/0.1% SDS at RT, then 3x 15 minutes in 0.1x SSC/0.1% SDS at 65 degrees C.
- Wrap blot in plastic wrap and place in a storage phosopho cassette.
- Image ~24 hours later, then lay down again for 7-10 day before imaging again.
We have been trying this for a month with this particular genotype and have had no luck. Attached is an example of what we've mostly been seeing with this protocol. Can anyone provide me with some advice on what I might be doing wrong and what I can do that might increase my chances of getting a clear signal? We are expecting bands at 16.85, 10.04, and 6.96.
Thanks so much for your help, excited to get some groupthink from a community of people with much more experience than I have!