Northern blot problem - help, plz (Nov/13/2005 )
I tried northern blot for my bacterial RNA, but I failed to get a signal.
The target RNA is larger than 3 kb.
RNA isolation, FA-gel electrophoresis, and transferring looked fine. I doubt the hybidization, but I need your advices to improve this step.
-The probe was prepared with asymmetric PCR method using Taq and 32P dCTP. The size of the probe is about 700 bp.
- Hybridization was done with the buffer with SSC, Denhardts, 0.5% SDS and formamide at 42oC for 18hrs. Do I have to increase the hybridization time?
- Washing was done with 2xSSC and 0.1% SDS, followed by 0.1X SSC and 0.1% SDS.
- I know there are commercial buffer system for Northern. Do you think it is worth to use it?
Thank you for your helpful advices in advance.
The target RNA is larger than 3 kb.
RNA isolation, FA-gel electrophoresis, and transferring looked fine. I doubt the hybidization, but I need your advices to improve this step.
-The probe was prepared with asymmetric PCR method using Taq and 32P dCTP. The size of the probe is about 700 bp.
- Hybridization was done with the buffer with SSC, Denhardts, 0.5% SDS and formamide at 42oC for 18hrs. Do I have to increase the hybridization time?
- Washing was done with 2xSSC and 0.1% SDS, followed by 0.1X SSC and 0.1% SDS.
- I know there are commercial buffer system for Northern. Do you think it is worth to use it?
Thank you for your helpful advices in advance.
Well to me everything looks pretty fine
some few possible problems are
1/ How good the labelling of the probe was ? You can always include a dot blot with diluted DNA spotted to have a positive control in your hybridisation
2/The stringency of the wash was maybe too high cause 0.1xSSC is laready quite stringent you can reduce to 0.5xSSC, 0.1%SDS and see
3/ The temperature of the washes also play a role in the stringency of the wash did you use 42°C or 65°C ?
4/ The transfert on the blot is maybe good but how sure are you that the transfert of the big RNA worked ? Did you monitored by checking the membrane ? Usually for big RNA transfert (viral RNA9 I use the alkaline tranfert and it worked fine.
Let us know
Pesji
Thank you, pesji.
I'll reduce the SSC concentration in washing step.
The temperature of washing is room temp for first wash(2x SSC), and 50 C for second wash(1x SSC).
I checked the transfer efficiency by staining the remaining gel with EtBr. No band appeared. After FA-GE, I incubated the gel in NaOH solution for 20min before blotting. Is it the same to the alkaline transfer?
I'll reduce the SSC concentration in washing step.
The temperature of washing is room temp for first wash(2x SSC), and 50 C for second wash(1x SSC).
I checked the transfer efficiency by staining the remaining gel with EtBr. No band appeared. After FA-GE, I incubated the gel in NaOH solution for 20min before blotting. Is it the same to the alkaline transfer?
Good for the washing step
As for the staining if you stain your gel before you can even vizualise the transferred RNA on the membrane (specially on Nylon)
For the alkaline transfert you should not only pretreat the gel but also transfer overnight with an alkaline buffer and not SSC buffer
Pesji