Hi everyone,
I want to perform DNA FISH on a few genomic locations for polyploidy detection. My lab is new to FISH and on a tight budget so instead of using BAC/plasmid, I'm generating ~10kb templates targeting a few genomic regions (for probe) from gDNA of my cell line.
My questions:
Must one use BAC for FISH probe? I designed a lot of primers for a few regions and am currently still at PCR stage (my PCR did not work yet, I'm using Pfu and HiFi Taq DNA Polymerase) but I'd like to know if PCR from cells' gDNA would work for nick translation and subsequently work for FISH.
Thanks for any advice!
Cheers
Hi Su
whatever the species, PCR will always work using the good conditions (purity of sample, specificity of primers to the target, PCR conditions and good use of chemistry).
the first thing to analyze is the specificity of your primers and to assess that you can use web tools as in UCSC (http://genome.ucsc.edu/cgi-bin/hgPcr) or NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). If the in iliac PCR doesn't work, you'll need to redesign your primers, it's far better.
fred
Hi Frederic Lepretre, Thank you for your reply.
I did check my primers with primer blast and make sure they are good for PCR as I always do. I forgot to mention I was doing PCR on centromeric regions that I chose with the least repetitive sequences. I wonder if centromeric regions are hard to be amplified? I suspect my gDNA source was not good as I could not see any band on agarose gel even with 200ng loaded. I got very good 260/280 though. Definitely, I will review my primers, thanks.
So, if PCR works, I can rely on my PCR product for FISH and I don't need BAC? Theoretically I think it will work but I just couldn't find supporting evidence and I don't understand why we must use BAC. Thanks!
Hi Su
ok fro the primers, well done.
seeing nothing on agarose gel even smears may show 2 things, poor quantity of DNA (take attention that phenol that is sometimes used for DNA preparation has the same comportment as DNA in some machines as the old bioanalyzer, not with the new that detects phenol) , or very quality of the samples.
BAC are mostly used because they're larger than PCR amplicons and therefore contain more incorporated fluorescence to be seen on slides. you'll need to design amplicons as larger it is possible for you and in your case of centromeric targeting due to high repetition sequences.
fred
Hi Frederic, thanks for your advice. I will re-extract gDNA from my cell line and probably use a kit this time.
For my centromeric region, I design primers targeting around 10kb for each amplicon. I'll redesign if when nick translation works but FISH signal is weak.
Cheers,
Su
Hi Su
10kb should be fine for FISH efficiency, I've tested 500bp long time ago and it worked, even with very weak signal.
all the best
fred
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