Matt:LabNotes/2017-5-4: Difference between revisions
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Program | Program | ||
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min | 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min | ||
{| | *Average Ct values | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
| | |- style="font-size:11pt" valign="bottom" | ||
| align=" | | align="right" width="51" height="14" | | ||
| | | width="51" | DNA | ||
| | | width="51" | RNA | ||
| width="51" | NTC | |||
| | |||
|- style="font-size:11pt" valign="bottom" | |||
|- | | height="14" | Ampligase | ||
| align="right" | 13.6666666666667 | |||
| N/A | |||
| align="right" | 27.1 | |||
| | |||
| align=" | |||
| | |||
| | |||
|- style="font-size:11pt" valign="bottom" | |||
| height="14" | SplintR | |||
| align="right" | 12.1366666666667 | |||
| align="right" | 12.0733333333333 | |||
| align="right" | 12.45 | |||
|- style="font-size:11pt" valign="bottom" | |||
| height="14" | SplintR 20% Formamide | |||
| align="right" | 19.1666666666667 | |||
| align="right" | 18.9333333333333 | |||
| align="right" | 17.58 | |||
|} | |} | ||
Revision as of 02:35, 12 July 2017
Agi15k_Feb2017 V4 in vitro Capture
Calculate Probes Needed
V4
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
- 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
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
- 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 | 13.6666666666667 | N/A | 27.1 |
SplintR | 12.1366666666667 | 12.0733333333333 | 12.45 |
SplintR 20% Formamide | 19.1666666666667 | 18.9333333333333 | 17.58 |