Matt:LabNotes/2016-11-15: Difference between revisions

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(Created page with "=In vitro capture with CA12kOct2016 V4 SplintR Ligase Test 2= *gDNA and UHRR *SplintR and T4 DNA Ligase for HBRR ==Reference== *[[Matt:LabNotes/2016-9-9#Results|SplintR in vi...")
 
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===Sample Groups===
===Sample Groups===
*RNA template = (30ng or 150ng) Universal Human Reference RNA (UHRR 740000-41)
*RNA template = 100ng Universal Human Reference RNA (UHRR 740000-41)
*DNA template = 300ng gDNA 12878
*DNA template = 300ng gDNA 12878


#NTC - 8.19ng V4 - SplintR
#NTC - 9.9ng V4 - SplintR
#30ng RNA - 8.19ng V4 - SplintR
#150ng RNA - 9.9ng V4 - SplintR
#150ng RNA - 40.9ng V4 - T4
#150ng RNA - 9.9ng V4 - T4
#300ng gDNA - 16.38ng V4 - Ampligase
#160ng gDNA - 9.9ng V4 - Ampligase


==Experiment==
==Experiment==
<!--
 
===V4 Padlock Probe Capture===
===V4 Padlock Probe Capture===
*Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc)
*Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc)
*DNA is 12878 80.3ng/ul
*DNA is 12878 80.3ng/ul
*V8 padlock probes: [[Matt:LabNotes/2015-1-21|874nM]]
*V4 padlock probes: *[[Matt:LabNotes/2016-11-2|20.7nM]]
**Add 5.2ul to samples 3 & 6 and then dilute remaining ~4ul to 19ul final volume
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
| width="51" height="14" | Sample #
| width="51" height="14" | Sample
| width="51" | RNA (50ng/ul)
| width="51" | RNA (50ng/ul)
| width="51" | DNA (80.3ng/ul)
| width="51" | DNA (80.3ng/ul)
| width="51" | V8 (42.7ng/ul)
| width="51" | V4
| width="51" | V8 (8.19ng/ul)
| width="51" | 10X Buffer
| width="51" | 10X SplintR Buffer (*=Ampligase)
| width="51" | H2O
| width="51" | H2O
| width="51" | Total
| width="51" | Total


|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 1
| height="14" | NTC
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 10
| align="right" | 1
| align="right" | 3
| align="right" | 3
| align="right" | 26
| align="right" | 17
| align="right" | 30
| align="right" | 30


|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 2
| height="14" | SplintR
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 5
| align="right" | 3
| align="right" | 3
| align="right" | 22
| align="right" | 30
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 10
| align="right" | 5.2
| align="right" | 0
| align="right" | 3
| align="right" | 21.8
| align="right" | 30
 
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 4
| align="right" | 0.6
| align="right" | 0
| align="right" | 0
| align="right" | 1
| align="right" | 3
| align="right" | 3
| align="right" | 25.4
| align="right" | 14
| align="right" | 30
| align="right" | 30


|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 5
| height="14" | T4
| align="right" | 3
| align="right" | 3
| align="right" | 0
| align="right" | 0
| align="right" | 0
| align="right" | 10
| align="right" | 5
| align="right" | 3
| align="right" | 19
| align="right" | 30
 
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 6
| align="right" | 16.4
| align="right" | 0
| align="right" | 5.2
| align="right" | 0
| align="right" | 3
| align="right" | 3
| align="right" | 5.4
| align="right" | 14
| align="right" | 30
| align="right" | 30


|- style="font-size:11pt"  valign="bottom"
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 7
| height="14" | Ampligase
| align="right" | 0
| align="right" | 3.8
| align="right" | 0
| align="right" | 0
| align="right" | 2
| align="right" | 2
| 3*
| align="right" | 10
| align="right" | 21.2
| align="right" | 30
 
|- style="font-size:11pt"  valign="bottom"
| align="right" height="14" | 8
| align="right" | 3
| align="right" | 3
| align="right" | 0
| align="right" | 15
| align="right" | 0
| align="right" | 5
| 3*
| align="right" | 19
| align="right" | 30
| align="right" | 30


|}
|}
*Added 50ul mineral oil on top
*Added 50ul mineral oil on top
'''Program'''<br>
'''Program'''<br>
* 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
* 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
* -> add 3ul Enzyme mix
*For Ampligase: -> 55C 20h -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold
*For SplintR and T4: -> 37C 15min -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold


*Add 3ul Enzyme mix prepared on ice!
*Add 3ul Enzyme mix prepared on ice!
**Samples 7-8: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
**Ampligase: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
**Samples 1-6: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O
**NTC & SplintR: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O
*Incubate at 37C for 15min
**T4:  
*94C for 2min
<!--
*Aliquot 10ul from each into separate strip of tubes and put at 4C for 1 hr until qPCR
*Enzyme digest template of remaining mixture 37C for 1 hr
**1ul Exo I/III (1:1) for DNA
**1ul RNaseH and Riboshredder (1:1) for RNA
***First add 1ul 5M NaCl (100nM NaCl or KCl for Riboshredder)
*94C for 2min
 
*Add 1ul Exo I/III (1:1) for unligated padlock probes to ALL tubes
*37C for 1 hr
*94C for 2min
===qPCR===
===qPCR===
====Primers====
====Primers====

Revision as of 23:55, 16 November 2016

In vitro capture with CA12kOct2016 V4 SplintR Ligase Test 2

  • gDNA and UHRR
  • SplintR and T4 DNA Ligase for HBRR

Reference

Experimental Outline

  1. CA12kOct2016 V4 Padlock Probe capture of gDNA and RNA
  2. Sequence to quantify probe efficiencies

Sample Groups

  • RNA template = 100ng Universal Human Reference RNA (UHRR 740000-41)
  • DNA template = 300ng gDNA 12878
  1. NTC - 9.9ng V4 - SplintR
  2. 150ng RNA - 9.9ng V4 - SplintR
  3. 150ng RNA - 9.9ng V4 - T4
  4. 160ng gDNA - 9.9ng V4 - Ampligase

Experiment

V4 Padlock Probe Capture

  • Dilute 1.5ul of 1ug/ul UHRR into 30ul total(50ng/ul final conc)
  • DNA is 12878 80.3ng/ul
  • V4 padlock probes: *20.7nM
Sample RNA (50ng/ul) DNA (80.3ng/ul) V4 10X Buffer H2O Total
NTC 0 0 10 3 17 30
SplintR 3 0 10 3 14 30
T4 3 0 10 3 14 30
Ampligase 0 2 10 3 15 30
  • Added 50ul mineral oil on top

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • -> add 3ul Enzyme mix
  • For Ampligase: -> 55C 20h -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold
  • For SplintR and T4: -> 37C 15min -> 94C 2min -> add 2ul Exo I/III -> 37C 2h -> 94C 2min -> 4C hold
  • Add 3ul Enzyme mix prepared on ice!
    • Ampligase: 1ul Ampligase + 1ul 10X Ampligase Buffer + 8ul H2O
    • NTC & SplintR: 12ul SplintR + 2ul 10X SplintR Buffer + 6ul H2O
    • T4: