Pooling and diluting Nextera libraries
Hi all,
I made some Nextera libraries last week and wanted to pool 8 of them for sequencing. Here's the Qubit concentration and volume containing 2 nM for each given the equation 1ng/ul = 3nM (which should be accurate given my size distributions).
Qubit result (ng/uL) / Volume containing 2 nM
0.073 / 9.13
0.904 / 0.74
9.01 / 0.07
2.34 / 0.28
1.13 / 0.59
1.43 / 0.47
7.24 / 0.09
0.26 / 2.56
In order to pool the libraries, I was going to take the amount that equals 2 nM of library for each, pool that (in this case resulting in a total volume of 22.39 microliters) and use 5 microliters of that for denaturation and dilution (according to the protocol that begins on the bottom of page 9 of this document:
http://supportres.illumina.com/docum...15039740-c.pdf).
Does this sound logical? I realize this is basically a high-school chemistry question but I'm a bit mathophobic and want to be sure I'm doing this correctly.
Thanks!
Kevin
I made some Nextera libraries last week and wanted to pool 8 of them for sequencing. Here's the Qubit concentration and volume containing 2 nM for each given the equation 1ng/ul = 3nM (which should be accurate given my size distributions).
Qubit result (ng/uL) / Volume containing 2 nM
0.073 / 9.13
0.904 / 0.74
9.01 / 0.07
2.34 / 0.28
1.13 / 0.59
1.43 / 0.47
7.24 / 0.09
0.26 / 2.56
In order to pool the libraries, I was going to take the amount that equals 2 nM of library for each, pool that (in this case resulting in a total volume of 22.39 microliters) and use 5 microliters of that for denaturation and dilution (according to the protocol that begins on the bottom of page 9 of this document:
http://supportres.illumina.com/docum...15039740-c.pdf).
Does this sound logical? I realize this is basically a high-school chemistry question but I'm a bit mathophobic and want to be sure I'm doing this correctly.
Thanks!
Kevin