Matt:LabNotes/2015-1-21: Difference between revisions
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===Probes, Target and Ampligase Buffer Mix=== | ===Probes, Target and Ampligase Buffer Mix=== | ||
*gDNA: 12878 ( | *gDNA: 12878 (80.3ng/ul) | ||
*[[Matt:LabNotes/2015-1-12 | First strand cDNA from UHRR]] | *[[Matt:LabNotes/2015-1-12 | First strand cDNA from UHRR]] | ||
*[[Matt:LabNotes/2014-11-19#Final_Probes_to_Order:_Trim_to_12.2C000_probes | CA12k_Nov2014 Probes]] | *[[Matt:LabNotes/2014-11-19#Final_Probes_to_Order:_Trim_to_12.2C000_probes | CA12k_Nov2014 Probes]] | ||
**[[Matt:LabNotes/2015-1-5#TBU_Gel_Quantification | V4 (10.7 ng/ul)]] | **[[Matt:LabNotes/2015-1-5#TBU_Gel_Quantification | V4 (10.7 ng/ul)]] | ||
**[[Matt:LabNotes/2015-1-15#TBU_Gel_Quantification | V7 (39.2 ng/ul)]] | **[[Matt:LabNotes/2015-1-15#TBU_Gel_Quantification | V7 (39.2 ng/ul)]] | ||
***Dilute | ***Dilute 2ul into 4ul for easier more accurate pipetting | ||
{| {{table}} | {| {{table}} | ||
Line 65: | Line 65: | ||
| 2||V4 - cDNA||2.5||3.8||3||20.7||30 | | 2||V4 - cDNA||2.5||3.8||3||20.7||30 | ||
|- | |- | ||
| 3|| | | 3||V4 - NTC||2.5||0||3||24.5||30 | ||
|- | |- | ||
| 4||V7 - cDNA||1||15||3||11||30 | | 4||V7 - gDNA||1||15||3||11||30 | ||
|- | |||
| 5||V7 - cDNA||1||15||3||11||30 | |||
|- | |||
| 6||V7 - NTC||1||0||3||26||30 | |||
|} | |} | ||
Line 74: | Line 78: | ||
* -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) | * -> 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. | * -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. | ||
*'''FORGOT TO ADD MINERAL OIL;''' added mineral oil after adding 3ul Ampligase mix | |||
====AmpLigase enzyme mix==== | |||
{| class="wikitable" style="text-align:center;{{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Stock conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Prepare volume 30ul''' | |||
|- | |||
| AmpLigase||5||U/ul||0.5||U/ul||2.00 | |||
|- | |||
| 10x AmpLigase Buffer||10||x||1||x||2.00 | |||
|- | |||
| H2O||||||||||16.00 | |||
|- | |||
| Total||||||||||20.00 | |||
|} | |||
===Add Sequence Adapters PCR=== | |||
====Primers==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Primer''' | |||
| align="center" style="background:#f0f0f0;"|'''Sequence''' | |||
| align="center" style="background:#f0f0f0;"|'''Index #''' | |||
|- | |||
| ISB_CA_AF||AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG|| | |||
|- | |||
| ISB_CA_AR.T1||CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG||Indx1 | |||
|- | |||
| ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||Indx2 | |||
|- | |||
| ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3 | |||
|} | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Index''' | |||
| align="center" style="background:#f0f0f0;"|'''Forward Primer''' | |||
| align="center" style="background:#f0f0f0;"|'''Reverse Primer''' | |||
|- | |||
| 1||1||ISB_CA_AF||ISB_CA_AR.T1 | |||
|- | |||
| 2||2||ISB_CA_AF||ISB_CA_AR.T2 | |||
|- | |||
| 3||3||ISB_CA_AF||ISB_CA_AR.T3 | |||
|- | |||
| 4||1||ISB_CA_AF||ISB_CA_AR.T1 | |||
|- | |||
| 5||2||ISB_CA_AF||ISB_CA_AR.T2 | |||
|- | |||
| 6||3||ISB_CA_AF||ISB_CA_AR.T3 | |||
|} | |||
====PCR Test==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''6.5X Volume''' | |||
|- | |||
| Captured template||1||0 | |||
|- | |||
| 10uM Forward Primer||0.4||2.6 | |||
|- | |||
| 10uM Reverse Primer||0.4||0 | |||
|- | |||
| 2X KAPA SYBG MM||12.5||81.25 | |||
|- | |||
| H2O||10.7||69.55 | |||
|- | |||
| Total||25||153.4 | |||
|} | |||
*Aliquot 23.6ul from 6.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer | |||
Program | |||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min | |||
[[File:012314_CA12kNov14_CaptureSequence_test.JPG | 650px]] | |||
*Looks good; NTC are both negative so I won't amplify on next PCR | |||
====PCR==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | |||
| align="center" style="background:#f0f0f0;"|'''4.5X Volume''' | |||
|- | |||
| Captured template||12||0 | |||
|- | |||
| 10uM Forward Primer||2||9 | |||
|- | |||
| 10uM Reverse Primer||2||0 | |||
|- | |||
| 2X KAPA SYBG MM||50||225 | |||
|- | |||
| H2O||34||153 | |||
|- | |||
| Total||100||387 | |||
|} | |||
*Aliquot 86ul from 4.5X master mix and add 12ul captured template and 2ul corresponding reverse primer | |||
Program | |||
98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min | |||
[[File:012614_CA12kNov14_CaptureSequencePCR.JPG|650px]] | |||
*Bead purification with 1.5:1 Beads to amplicon volume ratio | |||
**Eluted with 50ul total for each sample | |||
===PAGE Quantification=== | |||
*Load 2ul of each sample + 2ul loading dye | |||
[[File:2015-01-28_CA12k_Nov2014_V4V7_SequenceQuant.jpg|650px]] | |||
*Sample1 (V4 - gDNA):40ng/ul | |||
*Sample2 (V4 - cDNA):41ng/ul | |||
*Sample4 (V7 - gDNA):10ng/ul | |||
*Sample5 (V7 - cDNA):12ng/ul | |||
[[Media:2015-01-28_CA12k_Nov2014_V4V7_SequenceQuant.xlsx | concentration calculations]] |
Latest revision as of 02:29, 31 January 2015
CA12k_Nov2014 V4 and V7 in vitro Capture[edit]
Calculate Probes Needed[edit]
V4[edit]
Probe:target | 1000:1 | ' |
Probe size | 3514 | 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 (3514, 150nt) | 1.71585106x10^8 | g/mol |
Amount Probe req'd | 26.4 | ng |
V7[edit]
Probe:target | 1000:1 | ' |
Probe size | 2486 | 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 (2486, 150nt) | 1.21388894x10^8 | g/mol |
Amount Probe req'd | 18.7 | ng |
Probes, Target and Ampligase Buffer Mix[edit]
- gDNA: 12878 (80.3ng/ul)
- First strand cDNA from UHRR
- CA12k_Nov2014 Probes
- V4 (10.7 ng/ul)
- V7 (39.2 ng/ul)
- Dilute 2ul into 4ul for easier more accurate pipetting
Sample # | Sample Description | Probes | Target | 10X Ampligase Buffer | H2O | Total |
1 | V4 - gDNA | 2.5 | 3.8 | 3 | 20.7 | 30 |
2 | V4 - cDNA | 2.5 | 3.8 | 3 | 20.7 | 30 |
3 | V4 - NTC | 2.5 | 0 | 3 | 24.5 | 30 |
4 | V7 - gDNA | 1 | 15 | 3 | 11 | 30 |
5 | V7 - cDNA | 1 | 15 | 3 | 11 | 30 |
6 | V7 - NTC | 1 | 0 | 3 | 26 | 30 |
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.
- FORGOT TO ADD MINERAL OIL; added mineral oil after adding 3ul Ampligase mix
AmpLigase enzyme mix[edit]
Components | Stock conc. | Unit | Final conc. | Unit | Prepare volume 30ul |
AmpLigase | 5 | U/ul | 0.5 | U/ul | 2.00 |
10x AmpLigase Buffer | 10 | x | 1 | x | 2.00 |
H2O | 16.00 | ||||
Total | 20.00 |
Add Sequence Adapters PCR[edit]
Primers[edit]
Primer | Sequence | Index # |
ISB_CA_AF | AATGATACGGCGACCACCGAGATCTACACGCCTGCATATCGGGAAGCTGAAG | |
ISB_CA_AR.T1 | CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTGCCTTCCCGATATCCGACGG | Indx1 |
ISB_CA_AR.T2 | CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG | Indx2 |
ISB_CA_AR.T3 | CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG | Indx3 |
Sample | Index | Forward Primer | Reverse Primer |
1 | 1 | ISB_CA_AF | ISB_CA_AR.T1 |
2 | 2 | ISB_CA_AF | ISB_CA_AR.T2 |
3 | 3 | ISB_CA_AF | ISB_CA_AR.T3 |
4 | 1 | ISB_CA_AF | ISB_CA_AR.T1 |
5 | 2 | ISB_CA_AF | ISB_CA_AR.T2 |
6 | 3 | ISB_CA_AF | ISB_CA_AR.T3 |
PCR Test[edit]
Components | 1X Volume | 6.5X Volume |
Captured template | 1 | 0 |
10uM Forward Primer | 0.4 | 2.6 |
10uM Reverse Primer | 0.4 | 0 |
2X KAPA SYBG MM | 12.5 | 81.25 |
H2O | 10.7 | 69.55 |
Total | 25 | 153.4 |
- Aliquot 23.6ul from 6.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
File:012314 CA12kNov14 CaptureSequence test.JPG
- Looks good; NTC are both negative so I won't amplify on next PCR
PCR[edit]
Components | 1X Volume | 4.5X Volume |
Captured template | 12 | 0 |
10uM Forward Primer | 2 | 9 |
10uM Reverse Primer | 2 | 0 |
2X KAPA SYBG MM | 50 | 225 |
H2O | 34 | 153 |
Total | 100 | 387 |
- Aliquot 86ul from 4.5X master mix and add 12ul captured template and 2ul corresponding reverse primer
Program 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
File:012614 CA12kNov14 CaptureSequencePCR.JPG
- Bead purification with 1.5:1 Beads to amplicon volume ratio
- Eluted with 50ul total for each sample
PAGE Quantification[edit]
- Load 2ul of each sample + 2ul loading dye
File:2015-01-28 CA12k Nov2014 V4V7 SequenceQuant.jpg
- Sample1 (V4 - gDNA):40ng/ul
- Sample2 (V4 - cDNA):41ng/ul
- Sample4 (V7 - gDNA):10ng/ul
- Sample5 (V7 - cDNA):12ng/ul