After the Nick Translation protocol (abbottmolecular) of my PCR product (325bp product), the electrophoresis data are not good. There have been tested two different periods of time as well two different concentrations of my PCR product.
Why do you think your nick translation is not efficient? What would you expect to see on the gel if it was highly efficient?
And a 325bp probe will hardly work for FISH, which kind of FISH experiments are you planning to do?
I know, this it not an answer to your question, but some further details will increase the chances that your problem may be be solved,
Not quite sure what you are trying to do, but if you are trying to generate a fluorescent probe, would it not be simpler to add fluorescent labelled dNTPS to your PCR reaction and generate a labelled probe that way. If you want a ss probe then take PCR product, dilute it (eg 10x) and do single primer extension for 10+ cycles with fluorescent dNTP tracers.
I'd like to label a PCR product which is going to be used as a FISH Probe. I think that in the gel I have to see a 325bp band and not a smear in this length. I have used labeled probes like this one, but with not success, so I though that it might be aproblem.
It looks to me like you're running your nick translation reaction for far too long. Using much longer plasmid-sized templates I've done this at 15C for 90 minutes, generating probes of 200-500 bp in length. A better alternative, as Ian suggests, is to incorporate your labelled nucleotide during the PCR (either fluorescently labelled or something like DIG-dUTP, which you can then detect with an antibody following FISH) - just replace a proportion of the corresponding unlabelled nucleotide (dTTP in the case of DIG-dUTP) with the labelled one and run the PCR as normal; compared with the unlabelled PCR, this will give a slightly larger product, confirming label incorporation. Some things you should bear in mind: (1) 325 bp is fine for a FISH probe, so you don't need to chop it up, meaning that nick translation is not necessary; and, (2) unless your target is repetitive in the genome, a 325-bp probe is unlikely to give you a detectable signal following FISH. So, as Sabine says, some more details on what you're trying to do might be useful...
The 325bp product is from a housekeeping gene. I tried with lower times and in 1h I got good results in agarose gel. I'd like to create probes for a repetitive sequence, however I thought to try first with a housekeeping gene (18SrRNA) generating a PCR product for this and labeling it with nick translation.
Panagiotis Apostolou "I'd like to label a PCR product which is going to be used as a FISH Probe. I think that in the gel I have to see a 325bp band and not a smear in this length."
This is incorrect! Basically, the concept of nick translation is to produce random nicks and the ends are filled-up with your labeled nucleotide; therefore, you must obtain smaller fragments and a smear on your gel.
I highly recommend that you first do some basic reading regarding the methods you want to employ.
And as I said your probe might be too small for FISH.
Let's take things from the beginning. I'd like to make FISH for a gene . So I thought to perform PCR for this gene (which size is about 5kb). Then to perform nick translation to this product and then perform FISH. Is this available? However because the PCR was not so good and because I had already primers for smaller fragment (325) I performed PCR and nick translation to this.
Nick translation will only generate small fragments of < 325 bp which may not be suitable for getting a good signal. Better to label the probe by incorporation of fluorescent dNTPs. You will get a better probe of defined size and probably greater fluorescence. If you do not have a larger fragment to label, you can generate a pool of several adjoining PCR fragments covering a larger sequence.
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