DIG pcr labeling and northern blot - (Apr/17/2008 )
I have several questions regarding northern blot using DIG.
1. Can I PCR-label cDNA probes using the following protocol?
1. H2O variable
2. 10X PCR Buffer 5 ul
3. MgCl2 1.5-2.5 mM
4. PCR Dig Labeling Mix 5 ul (200 nM dNTP= 2mMdATP, dCTP, dGTP each, 1.9 mM dTTP, 0.1mM digoxigenin-11-dUTP)
5. Primer 1 25 pmol
6. Primer 2 25 pmol
7. Taq DNA polymerase 2.5 unit
8. Template DNA variable
Total 50 ul
2. I read that the western blot apparatus can be used for semi-dry northern blotting if you use a "frame support" for agarose gels. Is it possible not to purchase these frames and make my own instead? Or just use the normal passive transfer.
3. How do I quantify gene expression from northern blot? Is it by using a quantifiable RNA ladder marker? But if my probe is cDNA and target is mRNA what can I use?
4. can i use random primed labeling of probes to do northern blot gene expression quantification?
Sorry, I've no clues about Northern, but I'm often using DIG-labelling protocols.
As long as I know, the PCR DIG-labelling kit is an exclusive patent owned by Roche.
They sell you pre-mixed PCR buffer (including MgCl2), labelled and unlabelled dNTPs stocks and enzyme mix.
Why are you trying to customize the mix?
Did you have troubles with probes synthesis? In case: what kind of troubles?
If you are trying to improve a tricky reaction, I can suggest you to:
* use 50% labelled nucleotides + 50% unlabelled nucleotides
* increase the cycle number from 30 to 40.
1) your protocol seems ok, but you may have to adjust digUTP conc, as Ila suggested. If you're not using the kit, you must be aware that not all the taqs incorporate well the digUTPs, so if yours doesn't work to have to try a different one.
2) in my lab some attempts were done to transfer RNA from agarose gel with a trans-blot. The gel always melted down a bit and transfer was bad. Maybe if you carfully adjust the current it may work, but I suggest capillarity transfer.
3) Northern quantification is relative. You can compare expression in different samples but not absolutely quantify. The fact that the probe is dna and the target rna doesn't affect the results, just the sensitivity that is lower than with an RNA probe.
4) you can use random priming, but the probes are usually less sensitive that with pcr labelling.
Regards, and good luck