Northern blot hybridization problems - (Jun/12/2006 )
Hello everyone,
I am having a lot of trouble with my Northern blots. The RNA was intact, the transfer was good (I stained the membrane with methylene blue) and my probes were hot. I used dsDNA probes made using RT-PCR and labelled them with the rediprime system from Amersham. All my probes are from 400 to 600bp long. As far as I can verify, I did everything by the book. So what could it be that's causing the hybridization to fail? Does anyone know any alternatives that I should try?
Please help. Thanks.
single strand DNA labelled by T4PNK may be an alternative.
Did you try both church and denhardt's buffers?
did you try less stringency during washes?
Did you try both church and denhardt's buffers?
did you try less stringency during washes?
Thanks for the advice. I have tried all - low, moderate and high stringency - washes. I haven't tried Church buffer ... would it make a big difference?
I think my next step is going to be the T4 kinase labeling. Can I use the same primer that I used to make my probes?? Thanks again.
You should work on dot blots rather than worrying about gels until you get the detection working. Just spot total RNA and an RNA control in serial dilutions onto a membrane and try detecting it. You can also verify that your probe detection is working by spotting serial diultions of the probe directly on the membrane. Only bother with the gel after you have the detection working. Wouldn't a ssDNA probe be more effective? Can't you make that easily with an asymmetric PCR reaction?
you can use the same primer.
I've obtained better results with church buffer than with denhardt's buffer.
Hi,
I am trying to get my DNA probe to work on the Northern Blot. The probe is specific to the gene whose mRNA expression I am trying to identify but the blot shows up nonspecific binding to the 28s and 18s rRNA bands.
The band size I am looking for is 1.8kb which is close to the 18srRNA band. How can I reduce the expression from the 28s and 18s rRNA bands and obtain my band of interest?
The probe count I have used is 1million cpm/ml and 0.8million cpm/ml.
Thanks.
you may consider the fact of an mRNA extraction kit
Ask for a sample test to a company and they probably xill give you the opportunity.