Arichard:Notebook/2013/February

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February[edit]

February 7[edit]

  • Diluted the first 32 of the 48 200 uM celseq RT primers to 3 different concentrations corresponding to 3 different RNA loadings:
RNA 1 ng 100 pg 10 pg
[RT primer] 32 pmol/ul 3.2 pmol/ul 0.32 pmol/ul
  • See reagents for exact layout

February 8[edit]

  • Processed first 16 samples from total RNA. Performed first strand RT and second strand synthesis. Pooled samples for EtOH precipitation and left in -80 C until I get the other 16 samples ready.
  • Sample layout is as follows:
Sample # RNA input Sample # RNA input Sample # RNA input Sample # RNA input
1 1 ng 9 100 pg 17 100 pg 25 10 pg
2 1 ng 10 100 pg 18 100 pg 26 10 pg
3 100 pg 11 100 pg 19 10 pg 27 10 pg
4 100 pg 12 100 pg 20 10 pg 28 10 pg
5 100 pg 13 100 pg 21 10 pg 29 10 pg
6 100 pg 14 100 pg 22 10 pg 30 10 pg
7 100 pg 15 NTC 23 10 pg 31 NTC
8 100 pg 16 NTC 24 10 pg 32 NTC

February 14[edit]

  • Processing second batch of 16 samples through second strand synthesis. I will end up with 2 EtOH precipitation reactions, each with 16 samples.
  • Do CS primer reaction and IVT tomorrow.

February 21[edit]

  • IVT reaction and Mg2+ fragmentation
  • Richard Szubin ran a bioanalyzer on for my samples (Andrew 7: #1-16, Andrew 14: #17-32).

File:Bioanalyzer 2 21 2013.pdf

February 28[edit]

  • Starting a new run with Vipul Bhargava's samlples: A3 (50 pg/ul) and A100 (50 pg/ul).
  • 9 samples total: 1 pg of A3-1, A3-2, and A100; 10 pg of A3-1, A3-2, and A100; 50 pg of A3-1, A3-2, and A100.
Vipul's samples '
1 A3-1, 1 pg
2 A3-2, 1 pg
3 A100, 1 pg
4 A3-1, 10 pg
5 A3-2, 10 pg
6 A100, 10 pg
7 A3-1, 50 pg
8 A3-2, 50 pg
9 A100, 50 pg
  • I will also have 16 samples of total RNA as follows:
Sample RNA (pg) Sample RNA (pg)
1 100 9 10
2 100 10 10
3 10 11 10
4 10 12 10
5 10 13 10
6 10 14 10
7 10 15 NTC
8 10 16 NTC
  • I will also run an NTC for IVT with a 5 ul rxn vol, as well as a 5 ul positive control rxn using 2 ul of template from Brandon.
  • In total, I will have 4 rxns going in to IVT: AR 2/28, VB 2/28, +, and -.
  • Instead of using Phusion Flash to add the T7 promoter and 5' AD, I am switching to Bst Large Fragment (no 3' to 5' exonuclease activity). After EtOH precipitation, I will resuspend the ds cDNA in 4.76 ul of H2O. I will then add 0.64 ul 10X NEB Buffer 2 and 0.5 ul of 10 pmol/ul T7 primer. The Tm for 50% is 69 degC and the Tm for 90% is 66 degC. I will denature for 2 min @ 95 degC, followed by annealing for 10 min @ 66 degC. I will then add 0.5 ul Bst and incubate for 30 min @ 65 degC, followed by 20 min @ 80 degC to heat inactivate.