Nextera mate pair kit substitutions - Covaris and Dynabeads

[pjuneja]

I'm trying the Nextera mate pair kit for the first time in a collaborator's lab. My time here is limited so unfortunately I have to make do with the consumables already in the lab. Does anyone have any recommendations for the following substitutions:

-Covaris microtube (6x16mm) (#520045) in place of recommended T6 glass tubes (#520031 and #520042)

-Dynabeads M-270 in place of recommended M-280

I have never used either of these components. Would I need to modify the protocol in any way to make these substitutions?

Any help would be greatly appreciated!

Punita

[pjuneja]

From the preliminary Bioanalyzer results, it seems to have worked just fine with these improvisations. I did end up with low yield though (2 nM). I used the 500bp shearing settings with 100uL of sample in each microtube. We're running the MiSeq so we should know the avg insert size shortly.

[hfaoro]

Hi Punita,
For covaris tube issue you can follow the specifications recommended by Covaris in this link: http://covarisinc.com/wp-content/uploads/pn_010158.pdf.

Please, can you answer me a question: How many tubes are there in the nextera mate pair box 1? I did't find the end-repair, A-tailing and PCR mix in my kit!
Thanks in advance

[ArciMol]

Hi hfaoro,

It seems my reply comes too late, but your Nextera Mate Pair should have a TruSeq DNA sample prep kit with it. That's where the end-repair, a-tailing and PCR mix are.

[lorendarith]

In the beginning we used the T6 tubes but I switched back to the smaller ones we use for small-insert libraries and that works even better, as it appears that the shearing force is more accurate.

The difference between M280 and M270 is their charge, and it seems M280 are designed to capture longer sequences whereas M270 for shorter more specific ones. I read somewhere that they usually recommend M270 when you want to capture very short biotinylated stretches.
I would go with M280 in this case because what fragments you bind in this step will determine your positional coverage of the genome. It would be bad (for the assembly?) if you were eliminating some sequences in this step.
Could also explain the lower yield...