I am using the NEBNext Ultra II Kit for library prep. This was my first time doing a library prep and we received a trial kit. I had four samples and I used Adapter 2 from the NEBNext Multiplex Oligos for my first run through the library prep and PCR. I have bioanalyzed the results and need to PCR the DNA more. However, I am out of Adapter 2 reagent. Would it be acceptable or even possible to PCR the samples with another Adapter (for example using Adapter 3 or Adapter 4)? If not, what steps should I take in order to PCR the DNA? Would I need to go back and redo the Adapter Ligation? As I mentioned before, this was my first time completing a library prep so any advice/suggestions is welcome. Thank you!
Ok.. I am not sure If I am going on the right direction to answer that question, so, refer to the protocol after that.
My answer is yes, if you are talking about the index you will use to pull your sample out of the pooled samples (after sequencing), there is no problem on using a different index, as long as you don't add another sample with the same adapter and pool them together (that will mix your samples in a way you can't distinguish what came from A and what came from B).
Remember, at least for Illumina sequencing, if you are running one lane with more than one sample, you have to pool the samples with multiple indexes. Also, refer to Illumina (if thats the case) to check which indexes can be pooled together for a better yield.
I hope I helped you on that. Please let me know If you need any more help. I've run over a hundred Illumina libraries, thats where my knowledge comes from.
Once you have ligated your adaptors, you shouold amplify your library with the library amplication primers.
These primers are complimentary to the adaptor sequences A and B(P).
If you want to barcode different samples in order to be able to pool your samples in one lane, you should not use the adaptors from the NEB kit, but instead use a barcode set.
These barcode sets contain the adaptor sequences as well, so also the amplification primer binding sites.
Hi,
I don't have any experience about Library prep. But, I think it may possible to do it.
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