[Dun]

Hi, Microgirl123

What kind of PCR system do you use for the 2nd PCR (barcoding)?

After that, do you follow Nextera XT protocol or TruSeq protocol?

thx


[QUOTE=microgirl123;115867]"Do you know if the randomized Ns listed in the two PCR approach are still required with the latest Miseq software release?"

[pmiguel]

Variable N's are not required any longer. Yes, you get the variable intensities, but the instrument software compensates.

--
Phillip

[microgirl123]

I'm using the Nextera adapter set so I just follow the instructions for the Nextera (not XT) PCR and cleanup. Then I've been denaturing with NaOH to load on the MiSeq. I've been having trouble with cluster density recently though and am thinking that for my run tomorrow I'm going to denature with NaOH and then heat denature.

[adrian]

Hi:
I have a question about adding Ns next to barcode.
Is it recommended now to have Ns next to barcode for efficiency and high quality reads?

P5-NNBarocdeNNN-Primer-----------------

-------PrimerNNBarcodeNN-P7

OR

P5-Barcode-Primer-----------
-----Primer-Barcode-P7

I head that having Ns next to barcode increases base quality.

Also how to remove Ns in the demultiplexing?

thanks

[adrian]

Any suggestions.
thanks