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.