150nt insert size, generating libraries, question!

[ZAAB]

Hi All:
I'm preparing libraries for the HiSeq...
We're generating a library of variants, and the whole construct is 143nt long...with 30 nt in the middle that are variable (lots of variants!).

Trying to adapt the TruSeq protocols to my application, I've already had to resort to using Qiagen Minelute columns instead of AmpPure (my size is below the threshold (all in the wash)).

I am planning on producing 1ug of DNA (via PCR, unfortunately) to then do end-repair, phosphorylation, ligation, etc...

Am I on the right track here...or should I assume loss after shearing? By 'loss', I mean that not all gDNA is at the correct size after shearing, but I know my PCR'd DNA is All the right size...or do I just go forward with 1ug of PCR'd DNA?

Also, since my final size is going to be smaller than suggested, should I alter amplification/extension conditions in the cluster station? I'm not running the machine, so what should I tell the tech?

Thanks!

[shurjo]

We're generating a library of variants, and the whole construct is 143nt long...with 30 nt in the middle that are variable (lots of variants!).

If all your contructs are the same size, why do you need to shear? In fact, if you used Taq for the PCR, you can go straight to adapter ligation afterwards.

Just my two cents.

[ZAAB]

Exactly! Yes, I am using Taq, and Yes, I do not need to shear...
Just interested in the amount of DNA from a 1ug shearing 'reaction'..what amount is of the correct size...80%? I'll have 100%, so I don't want to overload the downstream analysis...

Thanks for confirming why I don't need to do endrepair, etc.

Z

[HESmith]

All of the input DNA is present through the gel extraction step, so all of the reaction conditions are calculated for that amount. It's immaterial what fraction is of the desired size until the PCR amplification, and at that point the protocol has recommended guidelines based on the amount of fragment.

[ZAAB]

I guess I was worried about overloading the ligation reaction with insert. I am never convinced that all DNA is carried forward when there is a wash/Ampure step.
Thanks for all replies.

I ended up performing the A-tailing rxn again, even though its TAQ-amplified, as I know A's do 'fall off' from experience. And, that volume and enzyme mix is present in the ligation rxn, with no cleanup involved between those steps.

THanks!