southern low sensitivity - how can I improve the sensitivity of my probe? (Mar/26/2007 )
Hi everybody!
I am struggling with the sensitivity of my southern probe which is a biotinylated DNA. First I blotted my gDNA onto the membrane and added 4 dots (concentrations: 100ng/µl; 10; 1; 0.1). Then, I crosslinked it, applied the prehyb. solution (30% formamide; 1% SDS, 1 M NaCl, 6% PEG, salmon sperm DNA, H2O) for about 60 min. Hybridisation was performed over night (same solution but with probe of course). However, after low stringency wash (2x SSC, 0,5% SDS) at RT and high str. wash (0,2x SSC, 0,5% SDS) at 46°C and 50°C and 55°C respectively for 40 min and application of conjugate (streptavidin-HRP) I saw only dots for the conc. 100ng/µl and 10ng/µl but as expected no bands of my gDNA. Shouldn’t the probe also reveal lower concentrations? So, I think there is a problem with the sensitivity of my probe, or what do you think? Any suggestions are welcome urgently!
Thanks a lot for any help!
winston2
I am struggling with the sensitivity of my southern probe which is a biotinylated DNA. First I blotted my gDNA onto the membrane and added 4 dots (concentrations: 100ng/µl; 10; 1; 0.1). Then, I crosslinked it, applied the prehyb. solution (30% formamide; 1% SDS, 1 M NaCl, 6% PEG, salmon sperm DNA, H2O) for about 60 min. Hybridisation was performed over night (same solution but with probe of course). However, after low stringency wash (2x SSC, 0,5% SDS) at RT and high str. wash (0,2x SSC, 0,5% SDS) at 46°C and 50°C and 55°C respectively for 40 min and application of conjugate (streptavidin-HRP) I saw only dots for the conc. 100ng/µl and 10ng/µl but as expected no bands of my gDNA. Shouldn’t the probe also reveal lower concentrations? So, I think there is a problem with the sensitivity of my probe, or what do you think? Any suggestions are welcome urgently!
Thanks a lot for any help!
winston2
probably you can reduce washing timings, i do for 10 min twice and one wash for 15 min
this might help
probably you can reduce the timings of washing i do for 10 min twicw and third washing for 15 min
Thank you! This is a good idea...I will try it...(I guess you mean the low stringency wach and the high str. wash, respectively)
...do you use the same SDS and SSC concentration?
winston2