SureSelect Hyb#4 - SDS

[Squall]

Hi all,

Could anyone tell me the role of SDS (Hyb#4 buffer I'm assuming) in the hybridisation reaction of SureSelectXT?

It is causing us some problems and we were wondering how disastrous it would be to titrate it back, or do away with it altogether.

Thanks!

[nucacidhunter]

SDS destabilises bonds between probes and library fragments (one of hybridization stringency factors) thereby reduces non-specific hybridization. Omitting it from hyb reaction would result in lower on target capture.

[Squall]

Thanks nucacidhunter.

Do you think that it is a linear response? So if we reduced the SDS by 20, 40, 80% etc, that the reduction in on-target would increase accordingly? Or is the SDS present in excess in the reaction?

[nucacidhunter]

I have not seen any formula to calculate the effects. I do not think that it is linear. One way to determine the effect empirically would be to reduce %SDS to the highest that downstream application can tolerate. If off-target levels are high then one can increase hybridisation temperature by 2C to compensate for reduced SDS content. Google searching DNA hybridization kinetics or dynamics should retrieve some related papers.

[melinn21]

I am also experiencing issues with this. While our data seems to be coming out ok, I'm finding that if I heat and then cool the hyb buffer mix (as the protocol says to do if precipitation forms) I somehow lose volume and never have enough to add to the probes (even with 5% overage). It's very strange. If I measure the volume while its still warm I have the correct volume if I measure while RT it is not enough and the precipitate has essentially returned.

[Squall]

Thanks hunter for suggestions, will cycle them into the testing plans!