size of probes for northern blot and other questions - end labelling of 20 mer oligonucleotides (May/26/2006 )
i have to do northern blot of COX-2. and i have the 20 mer oligonucleotides which i am going to end label with gamma p32 ATP.
Now i am afraiding that if i will use 20 mer probe then it might give non-specific binding also.
1. Here i want to know that will 20 mer probes be sufficient for the COX-2 hybridisation.
2. upto how much time i should give the exposure to x-ray film, generally paper have written that 1-6 days at -80 degree temp.
3. when transferring the RNA from gel to membranes shd i remove the well portion if yes then what is its advantages.
4. what is meaning of high stringency buffer in the washing procedure.
5. if i had stained my blot with methylene blue to check the trnsfer then how can i remove this stain.
6. if there are any other precaution which i shd take then plz tell. i will be very much thankfull to u for this act of kindnees.
It's worth a try. I've seen 25 mer probe for Northern, but not 20 mer. But that does not mean it'll not work. You can try higher stringency wash after hybridization to wash off unspecific bindings. The hybridization condition should stay the same with the system you're using, unless there's an added tip on how to enhance specificity of hybridization.
>>2. upto how much time i should give the exposure to x-ray film, generally paper have written that 1-6 days at -80 degree temp.
You can actually test it yourself. E.g. you expose for a few hours or a day, then develop the film. If the signal is not as strong as you wanted, then you can estimate the days you need to get the strong signal you want. If it's already suffice, then just another day would do. Best thing about radioactivity is it's half-life is longer than non-radioactive detection system and -80C actually helps to preserve the signal on the film.
>>3. when transferring the RNA from gel to membranes shd i remove the well portion if yes then what is its advantages.
As you please. But I don't. I just orientate the agarose gel, well surface facing downward and then place the nylon membrane (marked to determine correct orientation) onto it. The outlay of the samples and ladder on the blot later will be the same as what you see in UV illuminated and photographed gel. CAVEAT: Bubbles in the inverted wells sometimes will migrate to the middle of your gel if you're not careful.
>>4. what is meaning of high stringency buffer in the washing procedure.
You'll have to read it up. I get confused sometimes but it's less salt higher stringency and high salt less stringent.
>>5. if i had stained my blot with methylene blue to check the trnsfer then how can i remove this stain.
I normally just stain the agarose gel, not the blot. But there's no point staining them actually because there's bound to be residual ribosomal RNAs in the gel and bound to have rRNA on the blots as well. Just make sure the blotting procedure is extended longer or overnight.
Im also having this problem trying to northern cox2 with my PCR primers, I found somewhere that you can use a 300 base pair probe so I used my cox2 RT-PCR product and that didnt work either.
At the moment Im just doing dot blots to make sure the probe is working, but nothing
Im going to try my intgrety of the rna, but it works with the RT-PCR, co I dont know why it wouldnt work for this
I cant find any general information on making northern probes, even the company that makes the ECL signalling and probe labeling solutions (non-radioactive) cant help
........its driving me mad..............
Perhaps I should chat to you pBS.
Ryan Pink, Cranfield Health, Cranfield University. UK