TruSight One - no library

[JSAcierno]

Hi everyone,
I'm running the TruSight One panel, and the samples look great on Qubit and Bioanalyzer after the 1st PCR. However, at the end of the process I am validating the library and there is nothing there.....well, maybe a TINY little bit, but nothing in terms of the proper amounts.

Any ideas? TechSupport is trying to help, and they say the thermocycler may be the problem. We use the SensoQuest or the Biometra. They are saying the hyb and capture steps are very sensitive, and they have only approved 3 thermocyclers for this use. They say this ramping down step is extremely important, but cannot tell me what specs to test any other PCR machines with. In essence, they are saying it's a guessing game, and to just try multiple machines if we can't buy the recommended cyclers. 1 of the 3 recommended cylcers is now discontinued, 1 of the tetrads is on backorder for 6-8 months, and the last one is extremely expensive.

Does anyone have any experience with non-recommended cyclers?

Thanks,
Jim

[mikebravo]

Hi Jim,

we are using the biometra machines (TPROFESSIONAL BASIC) to generate TS1 libraries and the are doing fine. You have to pay attention that your thermocycler is suitable for 100µl. Otherwise the temperature will not be correct in your fluids. As far as I know there are different blocks for the biometra machines. The basic block ist for up to 70µl while the basic xl is suitable for 100µl reaction volume.

http://www.biometra.de/index.php/thermoblock.html

Do you perform the first hybridazation overnight?

kind regards,

-mike-

[JSAcierno]

Hi Mike,

Thanks for the reply. We are not doing the first hyb overnight. We used the SensoQuest, and not the Biometra, and we got bad results (after full library prep, only 1.1 ng/ul). Illumina sent new oligos, and we tried the SensoQuest with the old and new oligos, and an Eppendorf Nexus with old and new oligos. Illumina Field Apps was here while we did the lab protocols and saw nothing wrong. All libraries failed again, but the Eppendorf was about 3 ng/ul. Illumina says we should have at least 10 ng/ul as a bare minimum, but more likely 20-30 or even 50 ng/ul. We're really at a loss here.......

Jim