Arichard:Notebook/bulk150210

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Revision as of 19:07, 10 February 2015 by >Andrew (→‎=Lysis and freezedown)
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February 10, 2015 bulk Nextera on 1-02 and 25-01

February 10, 2015 bulk nextera

Motivation: I want to test a range of SM59 dilutions with our new IDT-Nextera v2 duel index primers against Nextera v1 with single index primers using bulk samples from 1-02 (AD) and 25-01 (CTL), but I don't have enough of all samples to compare everything at once. Therefore, my plan is to first extract DNA samples from the tubes I have in excess (cerebellum) to compare SM59 dilutions to Nextera v1, and wait on the more precious tubes (cortex) to use once I have optimized the SM59 dilution.

Goal: First, extract and freeze DNA from 1000 nuclei bulk samples from 1-02 and 25-01 cerebellum sorted October 9, 2014. Second, create libraries with SM59 dilutions and Nextera v1 and compare gels for intensity and distribution (tightest around ~300). Third, use best SM59 dilution to generate libraries from cortex.

Samples

sort # on tube nuclei/ul ul/tube nuclei/sample # samples # tubes to pool ul/sample ul HLS
1-02 ctx full 1 120 3 1000 1 3 8.33 26.67
1-02 ctx hi 2 120 3 1000 1 3 8.33 26.67
1-02 ctx lo 3 78 10 1000 1 2 12.82 22.18
1-02 cbl full 4 274 10 1000 3 2 3.65 31.35
25-01 ctx full 5 283 20 1000 3 1 3.53 31.47
25-01 cbl full 6 232 10 1000 3 2 4.31 30.69

Protocol

Following protocol from January 22 up to Nextera

Lysis and freezedown

  1. Lyse in 35 ul 1X HLS at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
  2. 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
  3. Elute in 7 ul H2O