Noi/NOTES/2013-3-12
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- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-6
Capture set up (Matt's project)[edit]
- Set up capture in 30ul
NTC (no targets) 1-1. gDNA (12878) 2-1. Total RNA 3-1. ds cDNA no RNasA treatment 4-1. ds cDNA RNaseA treatment 1-2. gDNA (12878) 2-2. Total RNA 3-2. ds cDNA no RNasA treatment 4-2. ds cDNA RNaseA treatment
Probe calculation[edit]
Probe:target | 1000:1 | ' |
Probe size | 12,335 | probes |
Template | 300 | ng |
Human gDNA MW | 1.9500E+12 | g/mol (3E+09 * 650Da/bp+157.9Da) |
Human gDNA (300ng) | 1.5385E-19 | mol (300ng/1.9500E+12 g/mol) |
Probe required (1000:1) | 1.5385E-16 | mol (1.5385E-19mol x 1000) |
Probe MW (12,335 probes, 170nt) | 6.8151E+08 | g/mol (12335 probes * 170nt * 325Da/nt + 79Da) |
Amount probe required | 1.05E-07 | g (6.8151E+08 g/mol * 1.5385E-16mol) |
Amount probe required | 105.00 | ng |
- Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul
- Note:
- I showed Matt how to calculate probes and he did calculation, and the number he got agreed with this calculation
- Even the target amounts in each reaction are not the same, I used the same probe amount in every reaction based on gDNA calculation
Probes and AmpLigase Buffer Mix[edit]
Components | Volume (ul) | 9.5 rxn mix |
12K Insitu probes (22ng/ul) | 4.77 | 45.32 |
10X AmpLigase buffer | 3.00 | 28.50 |
Total | 7.77 | 73.82 |
Samples | 12K Insitu probes | 10X AmpLigase buffer | Conc. (ng/ul) | Targets (ul) | H2O (ul) | Total (ul) | Total amount in capture (ng) |
NTC | 4.77 | 3.00 | 0 | 0 | 22.23 | 30.00 | 0.00 |
1-1. gDNA (12878) | 4.77 | 3.00 | 45.10 | 6.65 | 15.58 | 30.00 | 299.92 |
2-1. Total RNA | 4.77 | 3.00 | 1000.00 | 1.00 | 21.23 | 30.00 | 1000.00 |
3-1. ds cDNA no RNasA treatment | 4.77 | 3.00 | 15.20 | 19.00 | 3.23 | 30.00 | 288.80 |
4-1. ds cDNA RNaseA treatment | 4.77 | 3.00 | 5.84 | 19.00 | 3.23 | 30.00 | 110.96 |
1-2. gDNA (12878) | 4.77 | 3.00 | 45.10 | 6.65 | 15.58 | 30.00 | 299.92 |
2-2. Total RNA | 4.77 | 3.00 | 1000.00 | 1.00 | 21.23 | 30.00 | 1000.00 |
3-2. ds cDNA no RNasA treatment | 4.77 | 3.00 | 10.20 | 19.00 | 3.23 | 30.00 | 193.80 |
4-2. ds cDNA RNaseA treatment | 4.77 | 3.00 | 7.77 | 19.00 | 3.23 | 30.00 | 147.63 |
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
- -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
AmpLigase enzyme mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 30ul |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 3.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 3.00 |
H2O | 24.00 | ||||
Total | 30.00 |