I am curious to know if anyone has had extensive evaporation issues with sureselect during the 24h hybridization. I don't see any posts on the forums and everyone seems to be able to get it working fine except me of course. We have tried multiple methods of incubation and I ran 24hr tests prior to doing it on my samples as advised by the protocol. When I ran my tests I didn't get evaporation but when I run actual samples I always got total evaporation except for one time where 2 of 3 samples evaporated and one worked. I have spoken to Agilent tech support at length to try and fix this problem. I have tried the following:
1. pcr plate with adhesive seal
2. pcr plate with strip caps across it as well as adhesive seal
3. pcr tubes with strip caps
We are using an Applied Biosystems pcr machine with heated lid. The plates, tubes, and seals are all Applied Biosystems brands (the ones that are rated for the instrument). I sealed the tubes with this black thingy that comes with the machine to make sure the lids are tight. I always use interior positions on the thermal cycler as edges have known issues. At this point I guess the only explanation may be operator error but I'm hoping someone can shed some light. The main issue that I have is that my test reactions worked but actual sample reactions didn't. One potential problem that I see is that the test reaction is 100% hybridization buffer but the actual sample reaction is only 44% reaction buffer so maybe it isn't such a good control.
1. pcr plate with adhesive seal
2. pcr plate with strip caps across it as well as adhesive seal
3. pcr tubes with strip caps
We are using an Applied Biosystems pcr machine with heated lid. The plates, tubes, and seals are all Applied Biosystems brands (the ones that are rated for the instrument). I sealed the tubes with this black thingy that comes with the machine to make sure the lids are tight. I always use interior positions on the thermal cycler as edges have known issues. At this point I guess the only explanation may be operator error but I'm hoping someone can shed some light. The main issue that I have is that my test reactions worked but actual sample reactions didn't. One potential problem that I see is that the test reaction is 100% hybridization buffer but the actual sample reaction is only 44% reaction buffer so maybe it isn't such a good control.
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