Matt:LabNotes/2016-6-24: Difference between revisions

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>Mzcai
(Created page with "==in vitro Capture with TB12k_Apr2016 V7== *Will capture gDNA from mouse embryonic fibroblasts (got from Andrew) ===Sample Groups=== #gDNA MEF (77ng/ul) #Negative Control ==...")
 
>Mzcai
 
(5 intermediate revisions by the same user not shown)
Line 26: Line 26:
| Amount Probe req'd||14.6||ng
| Amount Probe req'd||14.6||ng
|}
|}
*[[Matt:LabNotes/2016-6-20#Qubit_Quantification|nM]]
*[[Matt:LabNotes/2016-6-20#Qubit_ssDNA|374nM]] = 18.2ng/ul




Line 38: Line 38:
| align="center" style="background:#f0f0f0;"|'''Total'''
| align="center" style="background:#f0f0f0;"|'''Total'''
|-
|-
| 1||gDNA|| ||3.9||3|| ||30
| 1||gDNA||0.8||3.9||3||22.3||30
|-
|-
| 2||NTC|| ||0||3|| ||30
| 2||NTC||0.8||0||3||26.2||30
|}
|}


Line 50: Line 50:
* -> 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 (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
* -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
<!--
**Tiffany accidentally stopped reaction after 30min of Exo I/III digestion
**She let it run 2min at 94C and then put it in fridge
**Soon after, she called and I asked her to add 2ul of fresh Exo I/III and finish out the protocol with 37C 2h -> 94C 2min -> 4C hold
 
====AmpLigase enzyme mix====
====AmpLigase enzyme mix====
{| class="wikitable" style="text-align:center;{{table}} border = 1
{| class="wikitable" style="text-align:center;{{table}} border = 1
Line 60: Line 63:
| align="center" style="background:#f0f0f0;"|'''Prepare volume 30ul'''
| align="center" style="background:#f0f0f0;"|'''Prepare volume 30ul'''
|-
|-
| AmpLigase||5||U/ul||0.5||U/ul||2.00
| AmpLigase||5||U/ul||0.5||U/ul||1
|-
|-
| 10x AmpLigase Buffer||10||x||1||x||2.00
| 10x AmpLigase Buffer||10||x||1||x||1
|-
|-
| H2O||||||||||16.00
| H2O||||||||||8
|-
|-
| Total||||||||||20.00
| Total||||||||||10
|}
|}


===Add Sequence Adapters PCR===
===Add Sequence Adapters PCR===
Line 82: Line 84:
|-
|-
| ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||Indx2
| ISB_CA_AR.T2||CAAGCAGAAGACGGCATACGAGATACATCGCGGTCTGCCTTCCCGATATCCGACGG||Indx2
|-
| ISB_CA_AR.T3||CAAGCAGAAGACGGCATACGAGATGCCTAACGGTCTGCCTTCCCGATATCCGACGG||Indx3
|}
|}


Line 94: Line 94:
| gDNA||1||ISB_CA_AF||ISB_CA_AR.T1
| gDNA||1||ISB_CA_AF||ISB_CA_AR.T1
|-
|-
| cDNA||2||ISB_CA_AF||ISB_CA_AR.T2
| NTC||2||ISB_CA_AF||ISB_CA_AR.T2
|-
| NTC||3||ISB_CA_AF||ISB_CA_AR.T3
|}
|}


Line 103: Line 101:
| 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;"|'''2.5X Volume'''
|-
|-
| Captured template||1||0
| Captured template||1||0
|-
|-
| 10uM Forward Primer||0.4||1.8
| 10uM Forward Primer||0.4||1
|-
|-
| 10uM Reverse Primer||0.4||0
| 10uM Reverse Primer||0.4||0
|-
|-
| 2X KAPA SYBG MM||12.5||56.25
| 2X KAPA SYBG MM||12.5||31.25
|-
|-
| H2O||10.7||48.15
| H2O||10.7||26.75
|-
|-
| Total||25||106.2
| Total||25||62.5
|}
|}


Line 121: Line 119:
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21


[[File:TB12k_Apr2016_V4_Capture_PCR_test.JPG|450px]]
[[File:2016-06-27_TB12kApr2016_V7_intubecapture_testPCR.jpg|450px]]


====PCR====
====PCR====
Line 127: Line 125:
| 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;"|'''2.5X Volume'''
|-
|-
| Captured template||12||0
| Captured template||12||0
|-
|-
| 10uM Forward Primer||2||9
| 10uM Forward Primer||2||5
|-
|-
| 10uM Reverse Primer||2||0
| 10uM Reverse Primer||2||0
|-
|-
| 2X KAPA SYBG MM||50||225
| 2X KAPA SYBG MM||50||125
|-
|-
| H2O||34||153
| H2O||34||85
|-
|-
| Total||100||387
| Total||100||250
|}
|}
   Program
   Program
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x26 -> 72C 3min -> 4C hold
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min -> 4C hold
*Take gDNA out after 25 cycles
*Take cDNA out after 30 cycles


[[File:TB12k_Apr2016_V4_Capture_PCR.JPG|450px]]
[[File:2016-06-27_TB12kApr2016_V7_intubecapture_PCR.jpg|450px]]


*Bead purification with 1.5:1 Beads to amplicon volume ratio
*Bead purification with 0.8:1 Beads to amplicon volume ratio
**Eluted with 50ul total for each sample
**Eluted with 50ul total for each sample


===PAGE Check===
===PAGE Check===
*Load 2ul of each sample + 2ul loading dye
[[File:2016-06-13_TB12kApr2016_V4_intubecapture_gelcheck.jpg|250px]]
*Bead purification did not work try again with 0.8:1 bead:sample ratio


[[File:2016-06-13_TB12kApr2016_V4_intubecapture_gelcheckRnd2.jpg|250px]]
[[File:2016-06-28_TB12kApr16V7_CapturegDNA.jpg|250px]]


*Labeled tubes and put in "Non-quantified Sequencing Libraries"
*Labeled tubes and put in "Non-quantified Sequencing Libraries"
**Sample1: MC20160610_TB12kApr16_gDNA-1 (MC160610_TB12kApr_gDNA-1)
**Sample: MC20160628_TB12kApr16V7_gDNA-1 (MC160628_TB12kAprV7_gDNA-1)
**Sample2: MC20150610_TB12kApr16_cDNA-2 (MC160610_TB12kApr_cDNA-2)-->

Latest revision as of 00:51, 29 June 2016

in vitro Capture with TB12k_Apr2016 V7[edit]

  • Will capture gDNA from mouse embryonic fibroblasts (got from Andrew)

Sample Groups[edit]

  1. gDNA MEF (77ng/ul)
  2. Negative Control

gDNA[edit]

Probe:target 1000:1 '
Probe size 1808 probes
DNA template 300 ng
gDNA MW 1.8x10^12 g/mol
gDNA (300ng) 1.66x10^-19 mol
Probe (1000:1) 1.66x10^-16 mol
Probe MW (1808, 160nt) 8.7997168x10^7 g/mol
Amount Probe req'd 14.6 ng


Sample # Sample Description Probes Target 10X Ampligase Buffer H2O Total
1 gDNA 0.8 3.9 3 22.3 30
2 NTC 0.8 0 3 26.2 30
  • Add 40ul Mineral Oil on top

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
    • In BioRad Thermalcycler program says: -0.2C per cycle every 30sec
  • -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
  • -> 55 C 20h (actually 24h)-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
    • Tiffany accidentally stopped reaction after 30min of Exo I/III digestion
    • She let it run 2min at 94C and then put it in fridge
    • Soon after, she called and I asked her to add 2ul of fresh Exo I/III and finish out the protocol with 37C 2h -> 94C 2min -> 4C hold

AmpLigase enzyme mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 30ul
AmpLigase 5 U/ul 0.5 U/ul 1
10x AmpLigase Buffer 10 x 1 x 1
H2O 8
Total 10

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
Sample Index Forward Primer Reverse Primer
gDNA 1 ISB_CA_AF ISB_CA_AR.T1
NTC 2 ISB_CA_AF ISB_CA_AR.T2

PCR Test[edit]

Components 1X Volume 2.5X Volume
Captured template 1 0
10uM Forward Primer 0.4 1
10uM Reverse Primer 0.4 0
2X KAPA SYBG MM 12.5 31.25
H2O 10.7 26.75
Total 25 62.5
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x21

File:2016-06-27 TB12kApr2016 V7 intubecapture testPCR.jpg

PCR[edit]

Components 1X Volume 2.5X Volume
Captured template 12 0
10uM Forward Primer 2 5
10uM Reverse Primer 2 0
2X KAPA SYBG MM 50 125
H2O 34 85
Total 100 250
 Program
 98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x13 -> 72C 3min -> 4C hold

File:2016-06-27 TB12kApr2016 V7 intubecapture PCR.jpg

  • Bead purification with 0.8:1 Beads to amplicon volume ratio
    • Eluted with 50ul total for each sample

PAGE Check[edit]

File:2016-06-28 TB12kApr16V7 CapturegDNA.jpg

  • Labeled tubes and put in "Non-quantified Sequencing Libraries"
    • Sample: MC20160628_TB12kApr16V7_gDNA-1 (MC160628_TB12kAprV7_gDNA-1)