Just wondering if anyone has any experience with this. I am still fairly new to preparing NexteraXT libraries. I have had good luck with the bead based normalization and running pools on the MiSeq, but now I want to send some pools to a HiSeq core lab.
My HiSeq core wants pools to be around 10nM. Illumina recommends pooling the double stranded libraries from the CAN plate and then using that for the HiSeq (vs the single stranded libraries coming out of the bead based normalization). I used a Qubit to quantitate one of my first CAN plates and the concentrations are fairly low, in the 500-3000 pg/uL range. The average size is around 800bp on Bioanalyzer. I can pool them but the pool volume is going to be large and the concentration very low, so I need to concentrate the pools.
Illumina recommends SpeedVac of the samples without heat, but tech support warned me if I used the buffer in the kit for the elution into the CAN plate (and why wouldn't have I?), then the salts in the buffer will concentrate as well, so they recommend not reducing the volume by >50%. They say in the future I can elute with H20 instead of the buffer in the kit, but that doesn't help me now. Any other column methods, while possible, would run the risk of sample loss. Ethanol precipitation might be an option, but the quantities are so low, I fear being able to see a pellet or achieve success with that method.
I haven't sat down to do the math yet to figure out my theoretical volumes, but just wondering if anyone else had any experience or advice for running NexteraXT libraries on the HiSeq.
Best,
DB
My HiSeq core wants pools to be around 10nM. Illumina recommends pooling the double stranded libraries from the CAN plate and then using that for the HiSeq (vs the single stranded libraries coming out of the bead based normalization). I used a Qubit to quantitate one of my first CAN plates and the concentrations are fairly low, in the 500-3000 pg/uL range. The average size is around 800bp on Bioanalyzer. I can pool them but the pool volume is going to be large and the concentration very low, so I need to concentrate the pools.
Illumina recommends SpeedVac of the samples without heat, but tech support warned me if I used the buffer in the kit for the elution into the CAN plate (and why wouldn't have I?), then the salts in the buffer will concentrate as well, so they recommend not reducing the volume by >50%. They say in the future I can elute with H20 instead of the buffer in the kit, but that doesn't help me now. Any other column methods, while possible, would run the risk of sample loss. Ethanol precipitation might be an option, but the quantities are so low, I fear being able to see a pellet or achieve success with that method.
I haven't sat down to do the math yet to figure out my theoretical volumes, but just wondering if anyone else had any experience or advice for running NexteraXT libraries on the HiSeq.
Best,
DB
Comment