Matt:LabNotes/2014-9-25: Difference between revisions

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(Created page with "==Repeat Top48 RT Primer in vitro Validation after DNA removal from UHRR== *Must first remove dsDNA from UHRR since there was contamination [[Matt:LabNotes/2014-6-18 | last ti...")
 
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**Centrifuge at 10,000rcf for 30sec
**Centrifuge at 10,000rcf for 30sec
*Store at -20C
*Store at -20C
===Reverse Transcription & Second Strand Synthesis===
====First Strand Synthesis====
*Mix RNA in Primer and H2O
*Denature for 5 minutes at 70C
**Put on ice right after
*Add Reaction Mix and Enzyme
*Incubate for 5 min at 25C
*Incubate for 1 hour at 42C
*Inactivate enzyme for 5 min at 80C
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Random Primer Mix'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Oligo d(T)23 VN'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''FISSEQ_RT'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Top48 Hexamer'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''NTC'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''No Enzyme'''
| align="center" style="background:#f0f0f0;"|''''''
|-
| Components||Volume||Components||Volume||Components||Volume||Components||Volume||Components||Volume||Components||Volume
|-
| Purified UHRR||1||Purified UHRR||1||Purified UHRR||1||Purified UHRR||1||Purified UHRR||0||Purified UHRR||1
|-
| Random Primer Mix (60uM)||2||Oligo d(T)23 VN (50uM)||2||FISSEQ_RT (100uM)||1||Top48 Hexamer (100uM)||1||Random Primer Mix (60uM)||2||Random Primer Mix (60uM)||2
|-
| H2O||5||H2O||5||H2O||6||H2O||6||H2O||6||H2O||5
|-
| Denature Step||||Denature Step||||Denature Step||||Denature Step||||Denature Step||||Denature Step||
|-
| M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10||M-MuLV Reaction Mix||10
|-
| M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||M-MuLV Enzyme||2||H2O||2
|}
====Second Strand Synthesis====
*Add 48 ul H20
*Add 8ul 10x Second strand synthesis reaction buffer
*Add 4ul Second strand synthesis enzyme mix
*Mix by pipetting
*Incubate for 2.5 hours at 16C
====Purification & Measure Concentration====
*Qiagen PCR Purification
**Elute with 30ul


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Revision as of 18:06, 26 September 2014

Repeat Top48 RT Primer in vitro Validation after DNA removal from UHRR

  • Must first remove dsDNA from UHRR since there was contamination last time

Purify UHRR of DNA

  • Use Zymo DNA-Free RNA Kit
  • Sample: 4ul of UHRR (1.3 ug/ul)

DNase I Digest

Components Volume
UHRR (1.3ug/ul) 4
10X DNase I Buffer 5
DNase I 2
H2O 39
Total 50
  • Mix and incubate at 37C for 30min

Purify

  • Add 100ul RNA Binding Buffer and mix
  • Add 150ul 100% EtOH and mix
  • Transfer to Zymo-Spin IC Column in Collection Tube
    • Centrifuge at 13,000rcf for 1min and discard flow through
  • Add 400ul RNA Prep Buffer
    • Centrifuge at 13,000rcf for 1min and discard flow through
  • Add 800ul RNA Wash Buffer
    • Centrifuge at 13,000rcf for 30sec and discard flow through
  • Add 400ul RNA Wash Buffer
    • Centrifuge at 13,000rcf for 30sec and discard flow through
  • Centrifuge at 13,000rcf for 2min and discard flow through
  • Transfer column to 1.5ul tube and add 8ul H2O and let stand for 1min
    • Centrifuge at 10,000rcf for 30sec
  • Store at -20C

Reverse Transcription & Second Strand Synthesis

First Strand Synthesis

  • Mix RNA in Primer and H2O
  • Denature for 5 minutes at 70C
    • Put on ice right after
  • Add Reaction Mix and Enzyme
  • Incubate for 5 min at 25C
  • Incubate for 1 hour at 42C
  • Inactivate enzyme for 5 min at 80C
Random Primer Mix ' Oligo d(T)23 VN ' FISSEQ_RT ' Top48 Hexamer ' NTC ' No Enzyme '
Components Volume Components Volume Components Volume Components Volume Components Volume Components Volume
Purified UHRR 1 Purified UHRR 1 Purified UHRR 1 Purified UHRR 1 Purified UHRR 0 Purified UHRR 1
Random Primer Mix (60uM) 2 Oligo d(T)23 VN (50uM) 2 FISSEQ_RT (100uM) 1 Top48 Hexamer (100uM) 1 Random Primer Mix (60uM) 2 Random Primer Mix (60uM) 2
H2O 5 H2O 5 H2O 6 H2O 6 H2O 6 H2O 5
Denature Step Denature Step Denature Step Denature Step Denature Step Denature Step
M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10 M-MuLV Reaction Mix 10
M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 M-MuLV Enzyme 2 H2O 2

Second Strand Synthesis

  • Add 48 ul H20
  • Add 8ul 10x Second strand synthesis reaction buffer
  • Add 4ul Second strand synthesis enzyme mix
  • Mix by pipetting
  • Incubate for 2.5 hours at 16C

Purification & Measure Concentration

  • Qiagen PCR Purification
    • Elute with 30ul