Matt:LabNotes/2017-5-4
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Agi15k_Feb2017 V4 in vitro Capture[edit]
Calculate Probes Needed[edit]
V4[edit]
Probe:target | 1000:1 | ' |
Probe size | 4998 | probes |
DNA templet | 300 | ng |
gDNA MW | 1.95x10^12 | g/mol |
gDNA (300ng) | 1.5385x10^-19 | mol |
Probe (1000:1) | 1.5385x10^-16 | mol |
Probe MW (4998, 157nt) | 2.387x10^8 | g/mol |
Amount Probe req'd | 36.7 | ng |
Probes, Target and Ampligase Buffer Mix[edit]
- gDNA: 12878 (80.3ng/ul)
- UHRR: 740000-41 (1ug/ul)
- Agi15k_Feb2017 Probe Design
- V4 Probe Production (10.3 ng/ul)
Sample | Condition | V4 (10.3ng/ul) | 12878 DNA (80.3ng/ul) | UHRR (1ug/ul) | Ampligase Buffer | SplintR Buffer | Formamide | H2O | Total |
1 | Ampligase DNA | 3.6 | 3.8 | 0 | 3 | 0 | 0 | 19.6 | 30 |
2 | Ampligase NTC | 3.6 | 0 | 0 | 3 | 0 | 0 | 23.4 | 30 |
3 | SplintR DNA | 3.6 | 3.8 | 0 | 0 | 3 | 0 | 19.6 | 30 |
4 | SplintR RNA | 3.6 | 0 | 1 | 0 | 3 | 0 | 22.4 | 30 |
5 | SplintR NTC | 3.6 | 0 | 0 | 0 | 3 | 0 | 23.4 | 30 |
6 | SplintR 20% formamide DNA | 3.6 | 3.8 | 0 | 0 | 3 | 6 | 13.6 | 30 |
7 | SplintR 20% formamide RNA | 3.6 | 0 | 1 | 0 | 3 | 6 | 16.4 | 30 |
8 | SplintR 20% formamide NTC | 3.6 | 0 | 0 | 0 | 3 | 6 | 17.4 | 30 |
Program
- 1,2,3,6: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
- 4,5,7,8: 55C 18h
- Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- SplintR: add 3ul SplintR enzyme mix (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O)
- Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
- SplintR: -> 37 C 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
- Zymo columns
- Elute 10ul
- Sample 2 was only able to pipette 20ul instead of 30ul of the ligation reaction so theoretically has 33% lower conc after elution from column
AmpLigase enzyme mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 10ul |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 1.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 1.00 |
H2O | 8.00 | ||||
Total | 10.00 |
Quantify[edit]
- On 5/18/2017
- qPCR all 8 samples 1ul each with triplicates
Components | 1X Volume | 25X Volume |
Captured template | 1 | 0 |
10uM ISB_CA_AF | 0.4 | 10 |
10uM ISB_CA_AR.T2 | 0.4 | 10 |
2X KAPA SYBG MM | 12.5 | 312.5 |
H2O | 10.7 | 267.5 |
Total | 25 | 600 |
- Aliquot 24ul from 24X master mix and add 1ul captured template
Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
- Average Ct values
DNA | RNA | NTC | |
Ampligase | 13.7 | N/A | 27.1 |
SplintR | 12.1 | 12.1 | 12.5 |
SplintR 20% Formamide | 19.2 | 18.9 | 17.6 |
Conclusion[edit]
- Accidentally chose 20% Formamide, should have been lower
- Odd that in 20% Formamide NTC had more captured padlock probes than the EXACT SAME conditions plus DNA or RNA