Dot Blot Hybridization using DIG-labeled probes - Help needed! (Nov/13/2009 )

Hello everyone, i have the following problem:

I isolated gDNA, amplified 3 target genes via PCR, performed a PCR-clean up, diluted my pcr products, applied 1.5 µl of my dilutions on a positively charged nylon membrane, UV-crosslinked, etc (everything according to the manual of DIG High Prime DNA labeling and Detection Starter Kit I, Roche). This worked fine for the pcr products after NBT/BCIP coloration: spot intensities decreased with increasing dilutions.

But: I wanted to do the same using gDNA (in comparison to the pcr products). I had 8 (very simple) dilutions. result: first 3 spots were visible and they had all the same intensity. Although I doubted it was a problem of dilution, I repeated the whole procedure with new dilutions, and again: pcr dilution row was fine whereas gDNA dilution row showed same intensities

any suggestions what the problem might be? I have no plausible explanation for this...

regards

Your genomic DNA may be sufficiently viscous and stringy that the dilutions are not really dilutions. Try reducing the viscosity of your genomic DNA by shearing (expression quickly through small diameter needles, vortexing vigorously with glass beads). Then make serial dilutions.

thank you for the hint, I´ll try that

Hi, everybody.
I would like to ask some questions about dot blot hybridization.
I have a lot of baculovirus samples. Some samples are mixed with two types of viruses: SeMNPV and AcMNPV. I would like to identify them which one is is SeMNPV and which one is AcMNPV. Can I use dot blot hybridization to identify both of them? Which characteristics and knowledge are need if I make dot blot hybridization? Please help me. I need about this information to continue my research. Thanks so much.
With best regards,
Moe.

What is a "lot?" If I had 100 samples, I would probably do QPCR on them. If I had 10,000 samples I would probably go to the trouble of optimizing a dot blot method. It will take you a while to get the probes and conditions correct for reliable dot blots. Do you need to?

please you can help me how you can make dilution for pcr product and how you can remove viscosity
thanks

Hi to each person
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