sureselect hybridization evaporation problems

[shawpa]

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.

[NextGenSeq]

I would seal the plates with two of the adhesive seals and use the compression pad in the cycler to press down on it. Even so I got evaporation. What I would do is add dH20 every 8 hours or so. We usually did 72 hybridizations for increased capture and enrichment.

[Michael.James.Clark]

You are not alone. This is an issue with the SureSelect platform. I believe the best solution we've had is the PCR plate/tubes with strip caps and just a lot of care when opening tubes in case liquid has adhered to the cap. Still, the volumes are so minute that we've had at least one run where we could see nothing left in the tube at one point.

[Cofactor Genomics]

I would recommend spinning the plate in a centrifuge, on a quick spin, prior to opening the caps. At times it looks like I might have had evaporation, however the liquid is simply in the cap. Hope this helps.

[odile]

I have the same problem and also use an Applied biosystem thermocycler (9700). Does anyone use some PCR tubes with cap locks? Are there better tubes than others? I'm not using Sure select but perform a similar hybridization capture.

Is it possible to use lab grade mineral oil or wax beads? Has anyone ever tried?

[Heisman]

One thing we've done that I think is helpful (although maybe it's placebo) is to place a piece of parafilm on top of strip caps but under the adhesive seal. Then the parafilm melts over the cap. We don't see bad evaporation issues as long as the strip caps are not crimped or anything like that.

[odile]

One thing we've done that I think is helpful (although maybe it's placebo) is to place a piece of parafilm on top of strip caps but under the adhesive seal. Then the parafilm melts over the cap. We don't see bad evaporation issues as long as the strip caps are not crimped or anything like that.

I think it's a good idea. Certainly worth the try since it can't hurt, right?

[Heisman]

I think it's a good idea. Certainly worth the try since it can't hurt, right?

Yep, just set up a water tube.

As I alluded to above, it's critical to make sure the strip cap is inserted into the tube without crimping in any way. That has been my biggest problem in the past.

[ahpolani]

One thing we've done that I think is helpful (although maybe it's placebo) is to place a piece of parafilm on top of strip caps but under the adhesive seal. Then the parafilm melts over the cap. We don't see bad evaporation issues as long as the strip caps are not crimped or anything like that.

Are you using then strip caps on a plate, parafilm on top and a film on top of that again?
Thanks!

[Heisman]

Are you using then strip caps on a plate, parafilm on top and a film on top of that again?
Thanks!

Yes, we use BioRad strip caps (can't remember the catalog number off the top of my head) that are flat and pretty deep so they snap into place. It is vital to put them in perpendicular to the plate, otherwise they can crimp a bit and you're screwed. I then take a small piece of parafilm and place it over the well(s). Then, I take two film covers and stagger them over the edges, rolling down each one individually. We rarely have any issues at all with evaporation.

[ECO]

We have great success with Eppendorf twin.tec 96 well plates, or knock offs, and sealing with 1 of the thick ABI Microamp seals (not the thin Optical ones), in a 9700. We've never gone to strip caps.