Hello
I want to check the presence of a gene on semen with FISH.
I don't have a large probe, so I need to design an oligonucleotide probe.
I heard that due to the weak signal, I should do first in situ PCR on the slide and then hybridize the probe.
Now I have some questions:
1. Is a probe size of 20-30 nucleotides enough?
2. Should be The Tm temperature around 70 c?
3. How do I determine the hybridization temperature? If the Tm of the secondary structures is 37 degrees, will there be any problem in hybridization the target gene?
4. If the probe is bind to other genes, will it cause problems?
because we are do in situ PCR , first.
5. What software do you use to design the probe? Do you use from primer design software?
I would appreciate your guidance and let me know your experiences, important points and protocols.
If you have an easier method, please suggest it.
Thanks
Hi Marjan,
I think I answered you on the wrong place.
I am not sure if I can help you, but I can try to give you some advice (but I can't guarantee this).
However, before I have some questions. So is it correct that you want to dedect a gene (DNA) with a DNA probe or do you want to dedect a transcript (RNA)?
It would also be nice to know somerhing about the fish species and the gene ( if you can give this information). How expirienced are you in doing PCR? Because if you want to do in situ PCR you should establish a working PCR before trying in situ PCR. Is there any reason why your probe size hast to be so small? In the next days I am really busy so it might take some time until I can andere you again.
KR Dörthe
Dörthe Andrea Kesper
Thank you.
I want to detect DNA with DNA probe.
PLP (chr X) & SRY (chr Y) in cattle (Bos taurus).
I have a good expirienced in PCR, real time and RFLP PCR, but I don't do in situ PCR.
Cost and ease of use are important to me.
If you know of another method, I would welcome it.
I would be grateful if you could guide me whenever you have the time.
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