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LacZ PCR


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#1 Douglas vasey

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Posted 08 October 2003 - 04:34 AM

I am currently screening transgenic mice for the LacZ gene. I am looking for an internal control to determine that DNA is present in the PCR reaction. Does anyone have any siggestions of house keeping genes that may be suited to this application ?

Thanks

#2 il0postino

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Posted 09 October 2003 - 08:23 PM

From your question i gather that you are just screening your mice to see whether or not they contain the transgene. I don't think that there should be any question if you added any genomic DNA to your tube. Its one of the few things you can actually see when you isolate it in molecular biology.
More importent probably is, how clean is it, and how much is it. These are the main factors that determine if your PCR reaction works (i mean that you can be sure that your LacZ gene will be amplified if it is present in the DNA).
So if you donot get any postive reactions you will probably need to do at least some of the following:
Measure the concentration of your genomic DNA. Then use the correct amount in the PCR reaction. Dilute a plasmid containing the LacZ gene in order to add about 1/1000.000 th the amount of it as compared to the genomic DNA to one of your reactions as a positive control.
If you do not get any signal try using less rather than more genomic DNA. In this way you will dilute the contaminants also making it more likely that your PCR will work.
If you really want to PCR some other gene to see if that works you can really take any primer pair. You see with genomic DNA your favourite "control" gene will be present in all cells/DNA samples. So you do not need a "house keeping gene". You need a primer pair that amplifies a particular piece of DNA. So open the freezer and see what mouse primers are there. Use a pair that is in coding sequence (this is least likely to be different between different mouse strains) and make sure that there is no intron within the amplified fragment. Also make sure it has a slightly different size as your LAcZ primers, That they have similar mt etc. If you add them to the same PCR reaction it can inhibbit the LacZ amplification or give other problems.

good luck




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