Raindance ligation before fragmentation

[glassfish]

Hello all,

I am doing a Raindance project. I tried NEB Quick Ligation™ Kit to concatenate the PCR products before fragmentation, but it doesn't seem to work very well for me. I still see some small PCR fragments after using the kit. Could someone share the information of what ligase might be better and the protocol? Thank you very much!

[h2karen]

We used the same NEB kit, and had similar problems originally when we tested the ligation with some test samples. Hence we did several things to improve the reaction. We combined the triplicates of our PCR products to increase the DNA concentration per sample, let the ligation for 45 mins at 20C, and did a second ligation after the first one (by adding more T4 ligase and MgCl2 to the first ligation reaction). When we checked the size on the bioanalyzer, we got a big peak at about 7000bp. Hope this helps!

[glassfish]

Thank you very much h2karen! I will try a second ligation. BTW, do you purify the PCR products again after ligation before sonication?

[h2karen]

You're welcome glassfish, I hope it works for you. And yes I did a purification before shearing for library preparation.

[epistatic]

I have been doing an end-repair and then ligation to ensure the ends were all completely blunt. This gives high molecular weight DNA after 15 minutes of ligation.

[glassfish]

Thanks epistatic! I tried to use PNK and do ligation twice, but still the results are not great. I will try end-repair.

[miroslav]

amplicon sequencing

Hello,
I'm new to this technology and have some issues with understanding various aspects. Can anyone decode the following question for me please? (An answer would be even better!)
How is non-redundancy handled with amplicon sequencing?
Your help will be most appreciated
Miroslav