Arichard:Notebook/bulk150210
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
- Lyse in 35 ul 1X HLS at RT for <5 min, followed by 35 ul of 8M guanidine HCl.
- 126 ul (1.8:1 ratio) Ampure XP, washed 2X with 80% EtOH
- Elute in 7 ul H2O