I'm setting up a PCR reaction for amplifying a sequence to design Southern blot DIG labelled probe, of size 450bp. For that I set up of two reaction one is control with normal dNTP's and other is with DIG labelled dUTP. I used 45oC annealing temperature for both the reactions. After running the 0.8% agarose gel I can see correct band in control well (where i used dNTP's without DIG labelling) but i didn't see any band in my sample of interest. Can anyone please suggest me is this problem is because of annealing temperature or for some other reason?
Digoxigenin is a fairly large size modification to the molecule and tends to destabilize the DNA duplex, which leads to a sizable proportion of fall-off products. Some of it depends on the relative molar ratio of DIG-dN versus its unlabeled counterpart. Lower proportions improve the abundance of full-length product but they also reduce the equivalent of specific-activity, leading to reduced signal in your final analysis of blots.
Run a small amount of amplified material on a gel, transfer it to a membrane and go through the detection of DIG using the antibody-enz conjugate. It is quite likely that you will see a smear starting from the actual size it should have been (or very close to it) extending to considerably smaller sizes. If you see about 40-50% of stain in close to the correct size, your product will work fine for the intended purpose. Remember that even a 200bp product represents the correct and sequence.
[I am assuming that the choice of primers may be limited otherwise you may have used a pair with higher Tm.]
Dhirend,
A better option is to produce single stranded cRNA probes. Because you will be using only a single strand and the hetero-dimers of cRNA and DNA are more stable, your sensitivity of detection will improve considerably. This will entail a little extra work, cloning your DNA in a vector with SP6 or T7 DNA-dependent RNA polymerase recognizing sequence and then liberalizing the plasmid at at an appropriate point that gives a suitable length of DIG labelled cRNA.
Hello Dhirend,
I´ve worked with were digoxigenin-labeled probes for Southern blot hybridisation in the past. I always labeled my DNA fragment myself using a kit by Roche (formerly distributred by Boehringer Mannheim) which was quite easily done. Maybe you could do the same (see below). It´s probably more expensive that PCR but it works well and the labeling procedure is fast.
Best wishes
Dirk
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