Dear Colleagues,
I am currently having difficulties with my PCR reaction from pre- stained PFA fixed zebrafish embryos. I had to stained the embryos and then needed to perform a PCR reaction which works each time and I routinely use. But unfortunately I do not get any band from any of my samples. Although I have tried different concentrations for the final DNA and ran 3 different reactions, non of the runs has worked!
The embryos were incubated in 4%PFA ON, goat serum+tritonX+antibody 2 times ON and washed in PBS then 1 night ON with goat serum+tritonX+ secondary antibody. After the staining the embryos were mounted on agar and analyzed with confocal microscope. Then from those embryos the DNA was isolated.
The DNA isolation is briefly; Lysis in NaOH with EDTA and followed by neutralization in Tris-HCl.
I have got around 25ng/ul DNA for each sample and use TAKARA AdvTaq for PCR.
Have you gotten any idea why my PCR did not work or more relevant why could not I get any amplification from my samples?
I have to use the DNA that I isolated, I do not have another option. Could any of you give me any suggestion?
Thank you very much!!!
best
Hi,
First, do you see unused primers on your gel ? If yes, they are fine. If not, may be they are degraded, so you should prepare new ones. I recommend the following links.
I hope it will work, Best Luck !
https://www.fws.gov/aah/PDF/PCRTS1_NoBand.pdf
https://www.researchgate.net/deref/https%3A%2F%2Fwww.neb.com%2Ftools-and-resources%2Ftroubleshooting-guides%2Fpcr-troubleshooting-guide
http://cshprotocols.cshlp.org/content/2009/4/pdb.ip66.full
Thank you very much for your answers. I do not see primers in my gel either. So I might need to prepare my primers again?
I can not amplify shorter fragments because I am going to use the amplicon for genotyping. I will need to use the DNA I isolated, since they are my only samples.
I have done quite a bit of genotyping after PFA and antibody using a proteinase K protocol but never tried the NaOH method on fixed tissue. Maybe NaOH does not work there? Pls let us know if prot k works better since most use NaOH now.
Here is the protocol:
Genotyping with Prot K
For DNA preps, larvae or fin clips were digested (10mM Tris, 2mM EDTA, 0.2% Triton X, 0.2mg/ml Proteinase K; pH 8.0) at 55°C overnight. The proteinase K was inactivated at 98°C for 15 minutes prior to PCR.
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