Hi, I am observing few backgrounds on metaphase spreads as if the probe is binding in "slides" as well as the metaphase spread and interphase. Any suggestion to get rid off the extra noise on slides would be appreciated. I was thinking of washing with 2xSSC at 72C twice in stead of once, any other suggestion? I am using only 1uL of probe with 9uL of buffer. Thanks!
Dear Tanjina
In my opinion you are using a lot of probe, we use 0.7ul of probe, 2.3 ul water and 7ul of buffer. With this method we have very good results on chromosomes. The signal is clear regardless of the quality of the metaphases. We spread the slides, leave them at room temperature 3 days and then apply the probe. The washing is normal, as describe the protocol of Vysis.
luis
Thanks Luis, just wondering how long do you hybridise after applying the probe? denature and hybridisation? Are your probes from Vysis? i am going to apply the homebrew probes soon. Was wondering whether you have tried metaphase spread with homebrew probes before?
Hi, We denature (codenaturalization) at 72 grades centigrades and after 20 hours of hybridization. We use only vysis probes.
after the second wash we wash the slides in water a few seconds.
good luck
luis
Hi, We denature at 72 grades centigrades during 3 minutes
Hello may I ask what type of sample are you processing?
Are your slides clean? Our post hybridization wash consist of washing in 0.4xSSC at 72oC for 2 minutes followed by 0.4xSSC at room temperature for a maximum 5 seconds for blood, amniotic fluid, and chorionic villus samples.
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