Matt:LabNotes/2014-9-25: Difference between revisions
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>Mzcai (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...") |
>Mzcai mNo edit summary |
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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