Problem with DIG High Prime DNA Labeling and Detection Starter Kit II - (Feb/12/2009 )

Hi to all!


I am trying southern blot for the detection of a single copy gene. My organism of interest is an insect. For the hybridization procedure i use DIG easy labeling and detection starter kit II.
My probe is a 900bp 3’RACE PCR product cloned into pGEM.The genomic sequence is not known but i do know the cDNA sequence.
After 3 unsuccessful attempts using 1μg,2.5μg and 5μg of total DNA i tried to find out which is the probe’s sensitivity. So i blotted several dilutions of digested 3’RACE plasmids.The dilutions i used were 12,1.2, 0.12 ,0.012, 0.0012 ng respectively. By using the standard protocols i get good signal till the 0.012ng dilution.
Translating this dilution into my insect’s gene copy number, i had to use 20μg of genomic DNA for taking good results………..So I did Blotting with 20μg. As control i used the above plasmid dilutions. All my controls worked fine also the marker gave a signal but still no band appeared in the gDNA area.
In general, as restriction enzymes i use HindIII, ECoRI and BclI.
Standard protocols for the transfer.
Fixation: Membranes into 6XSSC for 5 min, next UV cross-linking for 60sec into normal UV transilluminator and then baking for 3hrs into a 65 oC vacuum oven.
Hyb temp is 42oC, Hybridization over 18hrs,, high washes at RT, low washes at 68oC, blocking over 45min.
After the washes all the followed steps (Washing,Blocking,Antibody,Washing) are being performed at 42oC.
I think that blotting and fixing procedures are ok.I am not sure about the quality of my DNA..It seems to be good (Abs ratios are good, digestion is good).

Questions for you:
a.Should I change the probe ?
b.Do introns (if they exist) in the 3’ region could be subversive factors in the hybridization efficiency ?
c.How much clear should be the DNA ?Could high salt concentrations (came from 20XSSC) lower the hybridization efficiency ?
d.Should I change something in the fixation procedure?

I can’t understand what’s going wrong..

Plz HELP!!

I would spot your (uncut or cut) genomic DNA onto a membrane in serial dilutions and see if I could detect the gDNA with your probe. Only when that worked would I bother with the gel.

You appear to have more than one problem here:
1) You don't know if your Southerns are working due to a problem in the southern blot/hyb process, or
2) You don't know if your probe works on genomic DNA.

I will deal with 2) first... As you are using a cDNA as a probe and presumably this is composed of several exons rather than just one, there is a good chance that your probe is not binding properly to the genomic DNA as there is a lot of overhang/non-specific binding to be performed even if part of the probe binds to the genomic DNA. I would initially try and make a shorter probe and seeing if you can get binding with that - a bit of bioinfomatics searching may be able to give you a hint as to the intron/exon boundaries in the cDNA sequence which could help you design a better probe.

1) Presuming that you have read the DIG manual, your proceedure looks OK to me. I would try using only one of the restriction enzymes at a time, if you fragment the DNA too much you will never get a useable Southern. You could also try for a less stringent hybridisation and washing process, at least until you know it is working, and then increase the stringency.

I am putting my money on it being your probe that isn't working!