Matt:LabNotes/2017-5-4: Difference between revisions

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===Probes, Target and Ampligase Buffer Mix===
===Probes, Target and Ampligase Buffer Mix===
*gDNA: 12878 (80.3ng/ul)
*gDNA: 12878 (80.3ng/ul)
*UHRR: 740000-41 (1ug/ul)
*[[Matt:LabNotes/2017-2-8#Summary | Agi15k_Feb2017 Probe Design]]
*[[Matt:LabNotes/2017-2-8#Summary | Agi15k_Feb2017 Probe Design]]
*[[Matt:LabNotes/2017-4-12 | V4 Probe Production (10.3 ng/ul)]]
*[[Matt:LabNotes/2017-4-12 | V4 Probe Production (10.3 ng/ul)]]
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'''Program'''<br>
'''Program'''<br>
*gDNA: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
*1,2,3,6: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
*UHRR RNA: 55C 20h
*4,5,7,8: 55C 18h
*gDNA: 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)
*UHRR: add 3ul SplintR enzyme mix (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O)
*SplintR: add 3ul SplintR enzyme mix (13.5ul SplintR + 2.25ul 10X SplintR Buffer + 6.75ul H2O)
*gDNA: -> 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
*UHRR: -> 37 C 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
*SplintR: -> 37 C 15min -> 94C 10min -> add 2ul Exo I/III mix -> 37C 1h -> 94C 2 min -> 4C hold
*Zymo columns
*Zymo columns
**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====
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| Total||||||||||10.00
| Total||||||||||10.00
|}
|}
<!--
===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}}
===Quantify===
| align="center" style="background:#f0f0f0;"|'''Sample'''
*On 5/18/2017
| align="center" style="background:#f0f0f0;"|'''Index'''
#qPCR all 8 samples 1ul each with triplicates
| 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}}
{| {{table}}
| 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;"|'''6.5X Volume'''
| align="center" style="background:#f0f0f0;"|'''25X Volume'''
|-
|-
| Captured template||1||0
| Captured template||1||0
|-
|-
| 10uM Forward Primer||0.4||2.6
| 10uM ISB_CA_AF||0.4||10
|-
|-
| 10uM Reverse Primer||0.4||0
| 10uM ISB_CA_AR.T2||0.4||10
|-
|-
| 2X KAPA SYBG MM||12.5||81.25
| 2X KAPA SYBG MM||12.5||312.5
|-
|-
| H2O||10.7||69.55
| H2O||10.7||267.5
|-
|-
| Total||25||153.4
| Total||25||600
|}
|}
*Aliquot 23.6ul from 6.5X master mix and add 1ul captured template and 0.4ul corresponding reverse primer
*Aliquot 24ul from 24X master mix and add 1ul captured template


   Program
   Program
   98C 30s -> (98C 10s -> 52C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
   98C 1min -> (98C 10s -> 52C 20s -> 72C 20s)x30 -> 72C 3min


[[File:012314_CA12kNov14_CaptureSequence_test.JPG | 650px]]
*Average Ct values
*Looks good; NTC are both negative so I won't amplify on next PCR
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:11pt"  valign="bottom"
| align="right" width="51" height="14" | &nbsp;
| width="51" | DNA
| width="51" | RNA
| width="51" | NTC
 
|- style="font-size:11pt"  valign="bottom"
| height="14" | Ampligase
| align="right" | 13.7
| N/A
| align="right" | 27.1
 
|- style="font-size:11pt"  valign="bottom"
| height="14" | SplintR
| align="right" | 12.1
| align="right" | 12.1
| align="right" | 12.5
 
|- style="font-size:11pt"  valign="bottom"
| height="14" | SplintR 20% Formamide
| align="right" | 19.2
| align="right" | 18.9
| align="right" | 17.6


====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
[[Media:20170518_Agi15kFeb17_invitrocapture_SplintR20formamide.xlsx|Excel data]]
  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===
==Conclusion==
*Load 2ul of each sample + 2ul loading dye
*Accidentally chose 20% Formamide, should have been lower
[[File:2015-01-28_CA12k_Nov2014_V4V7_SequenceQuant.jpg|650px]]
*Odd that in 20% Formamide NTC had more captured padlock probes than the EXACT SAME conditions plus DNA or RNA
*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 02:44, 12 July 2017

Agi15k_Feb2017 V4 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 H2O Total
1 Ampligase DNA 3.6 3.8 0 3 0 0 19.6 30
2 Ampligase NTC 3.6 0 0 3 0 0 23.4 30
3 SplintR DNA 3.6 3.8 0 0 3 0 19.6 30
4 SplintR RNA 3.6 0 1 0 3 0 22.4 30
5 SplintR NTC 3.6 0 0 0 3 0 23.4 30
6 SplintR 20% formamide DNA 3.6 3.8 0 0 3 6 13.6 30
7 SplintR 20% formamide RNA 3.6 0 1 0 3 6 16.4 30
8 SplintR 20% formamide NTC 3.6 0 0 0 3 6 17.4 30

Program

  • 1,2,3,6: 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h
  • 4,5,7,8: 55C 18h
  • 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)
  • 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
  • Zymo columns
    • 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[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]

  • On 5/18/2017
  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
  • Average Ct values
  DNA RNA NTC
Ampligase 13.7 N/A 27.1
SplintR 12.1 12.1 12.5
SplintR 20% Formamide 19.2 18.9 17.6

Excel data

Conclusion[edit]

  • Accidentally chose 20% Formamide, should have been lower
  • Odd that in 20% Formamide NTC had more captured padlock probes than the EXACT SAME conditions plus DNA or RNA