DIG DNA PCR labeling problem - (Jul/01/2009 )
I used DIG DNA labeling kit from Roche to PCR a 250bp fragment from genomic DNA. However, I cannot see any band in EtBr staining gel. The samples with regular dNTP mix (same concentration) in the same program work fine.
Here is my PCR mix composition:
2.5ul 10X PCR Buffer (+Mg)
1.25ul Primers (10mM each)
1ul Genomic DNA as template(diluted)
2.5ul DIG DNA mix (10X) or 2.5ul regular dNTP mix (1mM each)
0.25ul Taq (Home-made)
H2O (up to 25ul)
Does anybody have that experience? Is there a fast way to check the DIG labeling efficiency rather than transfering onto N+ membrane.
Thanks very much.
Your protocol looks OK, and should have worked. You should be able to see the band on a gel stained with EtBr, and it should be larger than a band made with the same primers using ordinary dNTPs.
You should probably try using a high-fidelity polymerase to amplify your probe anyway, so as to have a more specific probe rather than one with potentially quite a few errors.
I tried Taq from Roche yesterday. However, one sample seems work. I still can't get any band from other two pair of primers. I can only see some primer-dimer band at bottom of the gel. No larger band appears. Is there some fast way to detect the DIG corporation?
Thanks for any suggestion.
If you can't see it on a gel, it probably hasn't worked. You could try dotting some of your labeled probe onto some membrane, cross-link it and then probe it with the anti-dig antibody and detect. It will probably take about 1-3 hours to do this in total.
bob1 on Jul 2 2009, 05:29 PM said:
There is no need to use a membrane. Buy the DIG test strips as it takes less than an hour to do...they also come with the random labeling kit, which you could also use...label the PCR fragment that you can amplify with random hexamers...maybe your DIG dNTP mix has degraded
If you are using the DIG system...don't cut corners...in general, when using Roche'sDIG system you have to use the materials they suggest to use...especially the easy hybe...works great.
After labeling, use HighPure PCR cleanup to remove unincorporated DIG dNTPs before quantification of the labeling rxn.
Since I brought the fresh kit last week and one of my sample works fine (sample 3), I don't think the DIG dNTP mix is degraded.
However, I run a PAGE gel today and transfer onto a Hybond membrane. After detection with anti-DIG antibody, I can clear see the clear band of sample 3, suggesting DIG is corporated in this sample. But the other two not-working samples showed a big smear with a condensed band in the cone region. Those DNAs are purified by Qiagen kit. Unfortunately, sample 3 is my control sample.
For the sample of interests, should I design another pair of primers to try it again
or should I try adding DMSO into PCR program?
Thanks for any suggestions.
Do your primers work for a normal non-DIG pcr? If not, then you should debug them with low expense pcr reactions. There is not much unique about the Roche dNTP mix -- You can just spike any normal Taq enzyme based PCR reaction with a little dUTP-DIG, which you can order from Roche quite cheaply.