Hello,
I am new here and to NGS. I prepared some multiplex DNA libraries with Illumina TrueSeq Kit. I submitted each sample/index at 10nM, to the facility that operated the sequencer. They told me that they used 1uL from each sample to make a 12uL total volume of pooled samples for one lane. This means each library was at 0.8nM. I got enough reads for that, and they used a GAII sequencer.
Now I have prepared samples for 8 lanes, multiplexing, in the same fashion. For a reason, we have to use other facility with a HiSeq 2000 sequencer. They asked me to have each library at 10nM. I don't have that concentration. Each library is 1.09nM for the lane with the least concentrated libraries. Other lanes are similar, maximum value is 3.38nM/per library (index). For the lane of 1.09nM I have 12 samples/indexes, so the dsDNA concentration for that lane is 13.08nM.
I think I will get enough coverage (72 cycles), but I will like to know if this concentration of dsDNA is enough for the machine to detect and amplify.
Please help me, I am so afraid to lose the work I have done for months :/
I put a lot of effort in my work, this is getting frustrating.
Thanks
C
I am new here and to NGS. I prepared some multiplex DNA libraries with Illumina TrueSeq Kit. I submitted each sample/index at 10nM, to the facility that operated the sequencer. They told me that they used 1uL from each sample to make a 12uL total volume of pooled samples for one lane. This means each library was at 0.8nM. I got enough reads for that, and they used a GAII sequencer.
Now I have prepared samples for 8 lanes, multiplexing, in the same fashion. For a reason, we have to use other facility with a HiSeq 2000 sequencer. They asked me to have each library at 10nM. I don't have that concentration. Each library is 1.09nM for the lane with the least concentrated libraries. Other lanes are similar, maximum value is 3.38nM/per library (index). For the lane of 1.09nM I have 12 samples/indexes, so the dsDNA concentration for that lane is 13.08nM.
I think I will get enough coverage (72 cycles), but I will like to know if this concentration of dsDNA is enough for the machine to detect and amplify.
Please help me, I am so afraid to lose the work I have done for months :/
I put a lot of effort in my work, this is getting frustrating.
Thanks
C
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