Failed Library Prep

[qsprengelmeyer]

Hello,

I used a NEBNext Ultra DNA library prep kit for Illumina sequencing. When I checked my samples on the Bioanalyzer none of them worked. The DNA concentration was too low to show up. This leads me to believe that either the adaptors failed to ligate, or there was a problem with the PCR step and they need to be amplified more. Anyone else experience this?

If I redo the adaptor ligation step, and the adaptors ligated properly the first time, will it ruin my samples?

Thanks,
Quentin

[nucacidhunter]

After USER enzyme step the ends of fragments are single stranded, so they cannot be ligated again. Lack of PCR amplification in addition to less efficient ligation and other factors could be also due to poor end repair and A tailing step.

[wdbestfly12]

Firstly, i don't know how you remove your redundant adaptors before PCR amplification steps. do you use the so-called :blind-cutting step or any other steps?
If using the blind-cutting, you may need to be very careful because the blind-cutting step can lead to low recovery of the ligation products significantly . you need slice the band within small scales. additionally,If your input sample is lower than 10 ng totally, you may hardly amplify the library with the classical 18 cycles of PCR amplification , If possible,you can add two or three another cycles and then you may see your library clearly. good luck

[IdahoRAD]

I have used this same kit, and I am really impressed with how it works. I've put in as little as 1 ng and have gotten out over 100 ng. Usually I have a 100-fold increase, with less than 1% being PCR duplicates (I was skeptical of such an increase at first!).

I think it is probably your adapters. We use our own adapters that don't require the USER enzyme. I believe they are called "Truseq" adapters from Illumina. I called NEB about using different adapters, and they said this was completely okay, so long as you adjust the annealing temp during the PCR.

Anyway, one thing you could do is to do a "test" PCR on a smaller volume of your template and do say, 20-24 cycles. Of course you won't sequence this, but if you are able to get brightness in the region you size selected for, at least you know the adapters are ligating on properly. Though, I think a BA would be more sensitive.