I am working on gene loci 3-D DNA FISH in suspension cells(primary lymphoid cells), but it's not working and I am not able to troubleshoot.I have tried to execute the experiment many times but I am unable to get the result. Followings are experimental details and queries:
Q1- How to isolate BAC DNA. I have isolated BAC DNA by Qiagen large construct kit but genomic DNA and RNA contamination are still coming.Is any special treatment required for isolating BAC DNA?
Q2- I am getting signal along with so much background so just help me out to reduce the background.
Q3-I am preparing probe by FISH Tag Kit(Invitrogen) by nick translation but I want to know how much concentration of probe DNA, cot1 DNA you used for precipitation of DNA and in how much volume of hybridization buffer and how many washes is required to get rid of the background? It would be nice if anybody can share the protocol with me.
Thanks, Shafqat Ali Khan. I will go through the protocol but I may mention that I am following this paper for my experiment.
https://link.springer.com/protocol/10.1007%2F978-1-60761-789-1_8#page-1.
I am also standardizing Immuno-RNA-DNA FISH and could help you after two weeks I think. But as ann answer of your third question, I would suggest using 10ng/ul probe and 100ng/ul Cot-1 DNA and dissolve it in 20ul of Hybridization buffer (this is for one reaction of FISH). This is not mentioned in the paper but I have got this information straight from the Lee lab. I will upload the detailed protocol tomorrow.
Hope this would be helpful.
thanks
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