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

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* -> 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.
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**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
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| 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]]
[[450px]]
 
<!--
====PCR====
====PCR====
{| {{table}}
{| {{table}}

Revision as of 22:27, 27 June 2016

in vitro Capture with TB12k_Apr2016 V7

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

Sample Groups

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

gDNA

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

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

Primers

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

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

450px