Automated vs. Manual fragment library preparation - need script!

[Brunda]

Hi,
We bought "48 fragment library construction kit" for Solid 5500, which is supposed to be used in automated systems like Tecan, but we don't have one so I am using this kit manually. As I do not have the right protocol, the A-tailing and adaptor ligation steps do not work. The A-tailing enzyme is different ( A-tailing enzyme II should be klenow fragment, this is what we have; A-tailing enzyme I contains taq polymerase)

I changed the temperature to 37°c (klenow's opt), but still poor results.

1. Did anyone solve this problem before?
2. can anyone provide me with tecan script so that I can find the right temperatures and incubation times?

Thanks in advance!

[JPC]

Hi Brunda, how can you tell which steps are failing? are you sure you're end polishing is okay, if not then all subsequent steps will fail.

For the manual protocol end polishing occurs at room temperature for 30 mins, the subsequent size selection process then removes the buffers and A tailing occurs at 68 degrees for 30 min. Have you tried that?

bw
JPC

[Brunda]

I do control multiNA run (similar to bioanalyzer) after size selection. Looks pretty much as it should, Yield ~30% of entry amount, 150-180 bp fragment lenght. How would you check if end polishing is OK?

For the manual protocol end polishing occurs at room temperature for 30 mins, the subsequent size selection process then removes the buffers and A tailing occurs at 68 degrees for 30 min. Have you tried that?

I did it like this for the first time and it yielded almost nothing - definitely not enough to enrich for exomes. There is also almost no amplification of library - which shows missing adaptors...

When I changed the temperature of A-tailing step to 37°c, then omitted the 72°c step in subsequent incubation (left nick translation to the following amplification), results were better but still not optimal

[JPC]

Yes your data suggests the adaptors are missing but it doesn't tell you which preceeding step is the problem, failure of; end polishing, A tailing or the adaptor ligation will all result in no adaptors.

If you run your A tailing at a low temperature then the manual suggests you risk having exonuclease activity from your polymerase.

How much starting material are you using? when we started out we used the maximum recommended 5ug but found that the libraries tended to fail, we've had much better results when we start with 1 to 3ug.

JPC

[Brunda]

I start with 1 ug DNA, End up with about 300 ng after size select (concentration 10 ng/ul) after amplification, I got approx. 3 ng/ul and a small peak on analyzer. So it seems that at least some adaptor ligation occurs, nevertheless the efficiency is bad.

After A-tailing, I heated the rxn to 72°c for 5 mins to inactivate A-tailing II enz. (in standard protocol, A-tailing I enz. is inactivated by cooldown, so I tried this), but i don't think this could have caused any degradation of the tails...
Other way to deactivate A-tailing II enz. is by EDTA (according to mate paired lib protocol), but I think it will deactivate T4 ligase as well...

I'll try to post the results from PCR and analyzer ASAP, but I don't have them on me now.

[Brunda]

You can take a look on control analyzer runs. Sample 1 Is the one with changed temperature. In Sample 2, i tried to replace A-tailing enzyme II with Taq polymerase (which should have the a-tailing activity) with no success.