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 (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O)
*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 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
*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
**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====
====AmpLigase enzyme mix====
Line 179: Line 180:


===Quantify===
===Quantify===
*On 5/18/2017
#qPCR all 8 samples 1ul each with triplicates
#qPCR all 8 samples 1ul each with triplicates


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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
<!--
 
 
[[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====
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| 2||2||ISB_CA_AF||ISB_CA_AR.T2
| 2||2||ISB_CA_AF||ISB_CA_AR.T2
|-
|-
| 3||3||ISB_CA_AF||ISB_CA_AR.T3
| 3||1||ISB_CA_AF||ISB_CA_AR.T1
|-
| 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
| 4||2||ISB_CA_AF||ISB_CA_AR.T2
|-
|-
| 10uM Reverse Primer||0.4||0
| 5||3||ISB_CA_AF||ISB_CA_AR.T3
|-
|-
| 2X KAPA SYBG MM||12.5||81.25
| 6||1||ISB_CA_AF||ISB_CA_AR.T1
|-
|-
| H2O||10.7||69.55
| 7||2||ISB_CA_AF||ISB_CA_AR.T2
|-
|-
| Total||25||153.4
| 8||3||ISB_CA_AF||ISB_CA_AR.T3
|}
|}
*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====
====PCR====
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| 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;"|'''4.5X Volume'''
| align="center" style="background:#f0f0f0;"|'''8.5X Volume'''
|-
|-
| Captured template||12||0
| Captured template||5||0
|-
|-
| 10uM Forward Primer||2||9
| 10uM Forward Primer||2||17
|-
|-
| 10uM Reverse Primer||2||0
| 10uM Reverse Primer||2||0
|-
|-
| 2X KAPA SYBG MM||50||225
| 2X KAPA SYBG MM||50||425
|-
|-
| H2O||34||153
| H2O||41||348.5
|-
|-
| Total||100||387
| Total||100||790.5
|}
|}
*Aliquot 86ul from 4.5X master mix and add 12ul captured template and 2ul corresponding reverse primer
 
*Aliquot 93ul from 8.5X master mix and add 5ul captured template and 2ul corresponding reverse primer
   Program
   Program
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min
   98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min
[[File:012614_CA12kNov14_CaptureSequencePCR.JPG|650px]]
 
*Bead purification with 1.5:1 Beads to amplicon volume ratio
*Sample 1: Stopped at 20 cycles
**Eluted with 50ul total for each sample
*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


===PAGE Quantification===
====For Sequencing====
*Load 2ul of each sample + 2ul loading dye
*MC20170619_Agi15kFeb2017_V4_AmpLigase_Indx1,2
[[File:2015-01-28_CA12k_Nov2014_V4V7_SequenceQuant.jpg|650px]]
*MC20170619_Agi15kFeb2017_V4_SplintRDMF_Indx1,2,3
*Sample1 (V4 - gDNA):40ng/ul
*MC20170619_Agi15kFeb2017_V4_SplintRForm_Indx1,2,3
*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 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]

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]

  1. 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