Matt:LabNotes/2017-6-19: Difference between revisions
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'''Program'''<br> | '''Program'''<br> | ||
*95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h | *95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h | ||
**Should have added RiboLock after reached 55C since it would be denatured at 95C | |||
*Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) | *Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer) | ||
*SplintR: add 3ul SplintR enzyme mix ( | *SplintR: add 3ul SplintR enzyme mix (10ul SplintR + 2ul 10X SplintR Buffer + 4ul Ribolock + 4ul H2O) | ||
*Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold | *Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold | ||
*SplintR: -> 37 C | *SplintR: -> 37 C 30min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold | ||
*Zymo columns | *Take 30ul and purify with Zymo columns | ||
**Left in 4C 1 hour after first column spin down | |||
**Elute 10ul | **Elute 10ul | ||
====AmpLigase enzyme mix==== | ====AmpLigase enzyme mix==== | ||
Line 180: | Line 180: | ||
===Quantify=== | ===Quantify=== | ||
#qPCR all 8 samples 1ul each with triplicates | #qPCR all 8 samples 1ul each with triplicates | ||
Line 204: | Line 203: | ||
Program | Program | ||
98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min | 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min | ||
[[File:20170622_Agi15kFeb17_invitrocapture_SplintR5formamide.PNG|350px]][[File:20170622_Agi15kFeb17_invitrocapture_SplintR5formamide_Ct.PNG|250px]] | |||
*[[Media:20170622_Agi15kFeb17_invitrocapture_SplintR5formamide.xlsx|Excel Data]] | |||
*No separation between DNA, RNA, and NTC samples for SplintR | |||
*Sequence results to see if there's no difference really | |||
**Stop Ampligase NTC at 28 cycles | |||
**Stop others at 19 cycles | |||
===Add Sequence Adapters PCR=== | ===Add Sequence Adapters PCR=== | ||
====Primers==== | ====Primers==== | ||
Line 231: | Line 239: | ||
| 2||2||ISB_CA_AF||ISB_CA_AR.T2 | | 2||2||ISB_CA_AF||ISB_CA_AR.T2 | ||
|- | |- | ||
| 3 | | 3||1||ISB_CA_AF||ISB_CA_AR.T1 | ||
|- | |- | ||
| | | 4||2||ISB_CA_AF||ISB_CA_AR.T2 | ||
|- | |- | ||
| | | 5||3||ISB_CA_AF||ISB_CA_AR.T3 | ||
|- | |- | ||
| | | 6||1||ISB_CA_AF||ISB_CA_AR.T1 | ||
|- | |- | ||
| | | 7||2||ISB_CA_AF||ISB_CA_AR.T2 | ||
|- | |- | ||
| | | 8||3||ISB_CA_AF||ISB_CA_AR.T3 | ||
|} | |} | ||
====PCR==== | ====PCR==== | ||
Line 269: | Line 256: | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
| align="center" style="background:#f0f0f0;"|'''1X Volume''' | | align="center" style="background:#f0f0f0;"|'''1X Volume''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''8.5X Volume''' | ||
|- | |- | ||
| Captured template|| | | Captured template||5||0 | ||
|- | |- | ||
| 10uM Forward Primer||2|| | | 10uM Forward Primer||2||17 | ||
|- | |- | ||
| 10uM Reverse Primer||2||0 | | 10uM Reverse Primer||2||0 | ||
|- | |- | ||
| 2X KAPA SYBG MM||50|| | | 2X KAPA SYBG MM||50||425 | ||
|- | |- | ||
| H2O|| | | H2O||41||348.5 | ||
|- | |- | ||
| Total||100|| | | Total||100||790.5 | ||
|} | |} | ||
*Aliquot | |||
*Aliquot 93ul from 8.5X master mix and add 5ul captured template and 2ul corresponding reverse primer | |||
Program | Program | ||
98C | 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min | ||
[[File: | |||
* | *Sample 1: Stopped at 20 cycles | ||
** | *Sample 2: Stopped at 28 cycles | ||
*Sample 3-5: Stopped at 18 cycles | |||
*Sample 6-8: Stopped at 17 cycles | |||
[[Media:20170626_Agi15kFeb17_invitrocapture_SplintR5formamide.xlsx|Excel data with PCR curve]] | |||
[[File:2017-07-10_Agi15kFeb17V4_invitroCapture_SeqLibrarySizeCheck.jpg|250px]] | |||
*Only Ampligase NTC band looks different, otherwise every sample has bands of expected size for captured padlock probes with sequencing adapters | |||
*Nanodrop: every sample was ~500ng/ul +/- 10ng/ul | |||
*Mix Samples 1&2, 3&4&5, 6&7&8 in equal ratios (20ul each) | |||
*Gel size selection 3 lanes | |||
**Load each well with 10ul mix (~5ug) + 10ul 6x loading buffer | |||
[[File:2017-06-27_Agi15kFeb17V4_invitroCapture_SeqLibrarySizeSelect.jpg|450px]] | |||
*Cut out band and shear through 0.5ul tube into 2.0ul tube | |||
*Add 450ul 1X TE Buffer and vortexed at 37C for 1hr | |||
*Centrifuged at 15,000 rpm for 3 min at RT | |||
*Transferred the clear spnt. to Nanosep columns and centrifuged at 15,000 rpm for 3 min | |||
*Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min | |||
*Transferred spnt to fresh 1.5 mL tube | |||
*Precipitated in 3 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2 | |||
*Vortexed and placed the 8 tubes at -80C 30min | |||
*Spun 8 tubes at 10,000rpm at 4C for 30min | |||
*Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min | |||
*Discard supernatant and let dry in hood for 10min | |||
*Resuspend each tube with 10ul and combine | |||
=== | ====For Sequencing==== | ||
* | *MC20170619_Agi15kFeb2017_V4_AmpLigase_Indx1,2 | ||
*MC20170619_Agi15kFeb2017_V4_SplintRDMF_Indx1,2,3 | |||
* | *MC20170619_Agi15kFeb2017_V4_SplintRForm_Indx1,2,3 | ||
* | |||
Latest revision as of 03:21, 12 July 2017
Agi15k_Feb2017 V4 + 5% formamide 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 or DMF | Ribolock 40U/ul | H2O | Total |
1 | Ampligase DNA | 3.6 | 3.8 | 0 | 3 | 0 | 0 | 0 | 19.6 | 30 |
2 | Ampligase NTC | 3.6 | 0 | 0 | 3 | 0 | 0 | 0 | 23.4 | 30 |
3 | SplintR 10% DMF DNA | 3.6 | 3.8 | 0 | 0 | 3 | 3 | 0 | 16.6 | 30 |
4 | SplintR 10% DMF RNA | 3.6 | 0 | 1 | 0 | 3 | 3 | 0.5 | 18.9 | 30 |
5 | SplintR 10% DMF NTC | 3.6 | 0 | 0 | 0 | 3 | 3 | 0 | 20.4 | 30 |
6 | SplintR 5% formamide DNA | 3.6 | 3.8 | 0 | 0 | 3 | 1.5 | 0 | 18.1 | 30 |
7 | SplintR 5% formamide RNA | 3.6 | 0 | 1 | 0 | 3 | 1.5 | 0.5 | 20.4 | 30 |
8 | SplintR 5% formamide NTC | 3.6 | 0 | 0 | 0 | 3 | 1.5 | 0 | 21.9 | 30 |
Program
- 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
- Should have added RiboLock after reached 55C since it would be denatured at 95C
- Ampligase: add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
- SplintR: add 3ul SplintR enzyme mix (10ul SplintR + 2ul 10X SplintR Buffer + 4ul Ribolock + 4ul H2O)
- Ampligase: -> 55 C 20h -> 94C 2min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2min -> 4C hold
- SplintR: -> 37 C 30min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
- Take 30ul and purify with Zymo columns
- Left in 4C 1 hour after first column spin down
- Elute 10ul
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]
- 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
File:20170622 Agi15kFeb17 invitrocapture SplintR5formamide.PNGFile:20170622 Agi15kFeb17 invitrocapture SplintR5formamide Ct.PNG
- No separation between DNA, RNA, and NTC samples for SplintR
- Sequence results to see if there's no difference really
- Stop Ampligase NTC at 28 cycles
- Stop others at 19 cycles
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 | 1 | ISB_CA_AF | ISB_CA_AR.T1 |
4 | 2 | ISB_CA_AF | ISB_CA_AR.T2 |
5 | 3 | ISB_CA_AF | ISB_CA_AR.T3 |
6 | 1 | ISB_CA_AF | ISB_CA_AR.T1 |
7 | 2 | ISB_CA_AF | ISB_CA_AR.T2 |
8 | 3 | ISB_CA_AF | ISB_CA_AR.T3 |
PCR[edit]
Components | 1X Volume | 8.5X Volume |
Captured template | 5 | 0 |
10uM Forward Primer | 2 | 17 |
10uM Reverse Primer | 2 | 0 |
2X KAPA SYBG MM | 50 | 425 |
H2O | 41 | 348.5 |
Total | 100 | 790.5 |
- Aliquot 93ul from 8.5X master mix and add 5ul captured template and 2ul corresponding reverse primer
Program 98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
- Sample 1: Stopped at 20 cycles
- Sample 2: Stopped at 28 cycles
- Sample 3-5: Stopped at 18 cycles
- Sample 6-8: Stopped at 17 cycles
Excel data with PCR curve File:2017-07-10 Agi15kFeb17V4 invitroCapture SeqLibrarySizeCheck.jpg
- Only Ampligase NTC band looks different, otherwise every sample has bands of expected size for captured padlock probes with sequencing adapters
- Nanodrop: every sample was ~500ng/ul +/- 10ng/ul
- Mix Samples 1&2, 3&4&5, 6&7&8 in equal ratios (20ul each)
- Gel size selection 3 lanes
- Load each well with 10ul mix (~5ug) + 10ul 6x loading buffer
File:2017-06-27 Agi15kFeb17V4 invitroCapture SeqLibrarySizeSelect.jpg
- Cut out band and shear through 0.5ul tube into 2.0ul tube
- Add 450ul 1X TE Buffer and vortexed at 37C for 1hr
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to fresh 1.5 mL tube
- Precipitated in 3 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C 30min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul and combine
For Sequencing[edit]
- MC20170619_Agi15kFeb2017_V4_AmpLigase_Indx1,2
- MC20170619_Agi15kFeb2017_V4_SplintRDMF_Indx1,2,3
- MC20170619_Agi15kFeb2017_V4_SplintRForm_Indx1,2,3