smear band in southern hybridization - (Jan/24/2006 )
Hello
I am facing the problem about southern hybridization by RI method. The result form RI is show that not have any band (only smear band) in each lane. In this experment, I want to make restiction map so I use 12 ug genomic fungal DNA and digest by several restriction enz. around 40 unit and digest o/n. after that I denature my gel by denature solution (1.5 M NaCl and 0.5 M NaOH) 1 time 30 min and neutralizing 2 time 10 min and transfer to membrane and do RI. The size of my probe is around 300 bp. Does the size sufficient to use as the probe?Hybridization temp is 65C and time is 2 hr. I think this point is not the problem. In my opinion, I think some system is not suitable.
please suggest me about your opinion.
Thank you
I am facing the problem about southern hybridization by RI method. The result form RI is show that not have any band (only smear band) in each lane. In this experment, I want to make restiction map so I use 12 ug genomic fungal DNA and digest by several restriction enz. around 40 unit and digest o/n. after that I denature my gel by denature solution (1.5 M NaCl and 0.5 M NaOH) 1 time 30 min and neutralizing 2 time 10 min and transfer to membrane and do RI. The size of my probe is around 300 bp. Does the size sufficient to use as the probe?Hybridization temp is 65C and time is 2 hr. I think this point is not the problem. In my opinion, I think some system is not suitable.
please suggest me about your opinion.
Thank you
Not an expert opinion but some experience can be shared through this reply. I don't know how RI work differently than conventional genomic/plasmid Southerns.
12 ug of Genomic DNA is too high quantity if you are going to use homologous probe.
Can you increase size of the probe? Say between 0.5 kb to 1.0kb. A 0.3 kb probe should not be a trouble.
Also your hybridization time might be insufficient.
Normally one would need to prehybridize membrane for at least 3 hours in case of genomic Southern and not less than 15 minutes for plasmid Southern. Also hybridization time should not be less than 8 hours in case of genomic while 3 hours in case of plasmid Southern.
If you had smear this might mean insufficient prehybridization led to nonspecific binding of the probe, paralogs on the chromosome for the probe you are using.
Also streak is seen if there is lot of salt leftover on the membrane, it happens with 20X SSC transfer and also 0.4M NaOH transfer.
If the streak is more from the bottom of the lane then it signifies degradation of genomic DNA
This can be avoided by changing buffers and water and also reducing digestion time from overnight to about 4 hours. If this is the case, try to use two controls, DNA in water+buffer no enzyme and DNA with water+enzyme no buffer. In frist control you should get intact DNA and second you might get intact, partial or star activity depending on enzyme you are using in digestion.
Good luck
Aru, Thank you so much 
for your suggestion. I will try to do following that. If I get the new result. I will inform you.
Thanks again