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 (80ng/ul)
*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 1ul into 2ul for easier more accurate pipetting
***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||V7 - gDNA||1||15||3||11||30
| 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]

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

concentration calculations