SureSelect Wash 2

[salyamoff]

Hi everyone! I had a failure with sureselect exome enrichment - no DNA in captured sample. I reread manual, then i read Faircloth (2013) "Target Enrichment of Illumina Libraries" and Holmberg (2005) "The biotin-streptavidin interaction can be reversibly broken using water at elevated temperatures" and i see that:
1) Samples need be washed 30 minutes total at 65C. (Manual)
2) Wash 2 buffer is 15 mM NaCl (Faircloth)
3) On this conditions streptavidin-biotin interaction will be broken with 100% chances. (Holmberg)
I don't think that SureSelect protocol is unreliable, but i think that my problem due to washing step.

[seqdavis]

Hello salyamoff,

We currently use Agilent's SureSelect XT and QXT protocols on a regular basis and have come across this a few times. Here are a few questions for your troubleshooting:

1. What was your target yield (ng) going into hybridization? We have found and the literature will tell you that one needs at least 250ng of pre-capture library going into hybridization. If this is not the case, you will have to adjust your bait ratio.

2. Did you perform the initial 95C denaturing step prior to the addition of your baits? If this doesn't occur, there are no single stranded molecules for the bait to anneal to therefore, no material will get 'pulled down.'

3. During your washing at 65C, did you make sure the beads were resuspended prior to the 10 minute incubation (which is done 3 separate times)?

4. Did you use water anywhere in the washing?

Take a look at these sections of your experiment and see if anything stands out. From my experience, this is where the majority of issues arise with the SureSelect protocols.

[salyamoff]

I repeated a enrichment and capture with another beads. Sequensing was done good. It looks like my dynabeads was depraved. But this beads succesfully captured biotenylated proteins recently. I think it because protein capture conditions aren't so extreme.