Hi, my Cre genotyping primers produce a single band if positive, and no band if negative, which can lead to false negatives.
Does anyone routinely genotype using some sort of internal positive control to show the PCR reaction worked well, template was good, etc?
thanks
Hello Dudley,
Unfortunately most Cre-lines are generated by a random integration of the cre-construct into the genome. Therefore a PCR on the unknown native gene is mostly not possible because of the unknown sequence. However, you can minimize the risk of false negative results within the cre-PCR by performing a PCR on the target (e.g. a deletion PCR).
Best,
Jan
Hi!
The answer is a little bit late, but maybe it´s helpful..
If I got you right you are only looking for a control for your PCR reaction, nothing specific..
We are using this protocoll from Qiagen, with the primers and protocoll mentioned there, but with normal gel electrophoresis.. works quite good and you can do both in one reaction..
https://www.qiagen.com/de/resources/resourcedetail?id=ba5d1a43-f6de-456e-a549-6d0488a4f764&lang=en
Best,
Martina
In order to minimize the false negative results, the following methods might be useful.
1. Design two pairs of primers specific targeting different Cre sequence areas, it may decrease the false negatives.
2. Run a Non template control (NTC) each time. If the NTC have bands, aerosol pollution of PCR products may exist in your lab. Try to remove it.
3. Select a High fidelity enzyme to avoid false negatives
Try the above three methods if the weak Cre band seen in the wild type mice.
Sheng Cao
Hi,
yes we also see sometimes weaker bands at approximately 100 bp.
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