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..
Edited by dimitri, 12 February 2009 - 01:11 PM.