Can someone help?
I have adapter dimmers or some other contaminant below 200 (probably 170bp). Ultimately, my goal is to remove the contaminates and pool the libraries.
My cDNA library stocks range between 125-348ng in 15uL each, and according to Nextra Lib Validation, that's 25-67nM/ uL (1ng/uL=3nM for a 500bp avg library size). Illumina TruSeq wants me to pool 10uL of each library normalized to 10nM.
My plan is to modify the protocol I used to get my stock libraries:
3.F.1. AMPure XP Purification - http://www.epibio.com/docs/default-s...t.pdf?sfvrsn=8
First, I will transfer between 3 and 8uL of stock libary into enough RNase-Free water to make 25uL, instead of the protocols 50uL.
Second, instead of using 1 volume of AMPure XP beads, I was going to use 0.9 volumes.
Continuing through the protocol, I will do the 200uL 80% EtOH washes, air dry and resuspend the beads in 20uL. Thus I hope to get 20uL of 10nM libraries.
Does this sound like it will work?
Also, I was wondering if anyone knows why using lower volumes of AMPure XP beads removes smaller species (<200bp?) Are these retained by the beads or do they evaporate with the EtOH, maybe?
Thanks for your help!
I have adapter dimmers or some other contaminant below 200 (probably 170bp). Ultimately, my goal is to remove the contaminates and pool the libraries.
My cDNA library stocks range between 125-348ng in 15uL each, and according to Nextra Lib Validation, that's 25-67nM/ uL (1ng/uL=3nM for a 500bp avg library size). Illumina TruSeq wants me to pool 10uL of each library normalized to 10nM.
My plan is to modify the protocol I used to get my stock libraries:
3.F.1. AMPure XP Purification - http://www.epibio.com/docs/default-s...t.pdf?sfvrsn=8
First, I will transfer between 3 and 8uL of stock libary into enough RNase-Free water to make 25uL, instead of the protocols 50uL.
Second, instead of using 1 volume of AMPure XP beads, I was going to use 0.9 volumes.
Continuing through the protocol, I will do the 200uL 80% EtOH washes, air dry and resuspend the beads in 20uL. Thus I hope to get 20uL of 10nM libraries.
Does this sound like it will work?
Also, I was wondering if anyone knows why using lower volumes of AMPure XP beads removes smaller species (<200bp?) Are these retained by the beads or do they evaporate with the EtOH, maybe?
Thanks for your help!
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