Matt:LabNotes/2014-6-5
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Top48 Hexamer RT Primer in vitro Validation[edit]
- Designed 48 Hexamer RT primers that only match mRNA sequences and not rRNA
- Do in vitro RT to make cDNA for sequencing
- Use sequencing results to validate that these 48 primers prime mRNA more than rRNA when compared to Random Hexamer currently used for FISSEQ
- Got RNA sample from Rui
- UHRR (~2ug/ul)
- Use Enzymatics M-MuLV RT for First Strand Synthesis
- Use NEB Second Strand cDNA synthesis for Second Strand
Procedure[edit]
- Mix the 200uM 48 primers in the 96-well plate in a 1.5mL tube
- Take 2ul from each well and then add 96ul H2O to make a 100uM Top48 primer mix
First Strand Synthesis[edit]
Component | Pos Ctrl | Neg Ctrl | Exp |
25mM dNTP | 1 | 1 | 1 |
~2ug/ul UHRR | 0.5 | 0.5 | 0.5 |
100uM RanHex Primer | 1 | 1 | 0 |
100uM Top48 Primer | 0 | 0 | 1 |
H2O | 6.5 | 6.5 | 6.5 |
- Heat reaction mix (above) to 65C for 5min
- Add 1ul 10X M-MuLV RT Buffer (total 10ul volume)
- Incubate 25C for 10min
- Add 1ul M-MuLV Reverse Transcriptase to Pos Ctrl and Exp ONLY (1ul H2O for Neg Ctrl)
- Incubate 42C for 60min
- Inactivate by heating to 85C for 10min
Second Strand Synthesis[edit]
- Add 57ul H2O
- Add 8ul 10X Second Strand Synthesis Buffer
- Add 4ul Second Strand Enzyme
- Mix by pipetting
- Incubate 16C for 2.5 hours
- Purify with Qiagen PCR Purification Kit
- Elute with 30ul H2O
Check Concentration[edit]
- With Nanodrop
- PosCtrl: 31.6 ng/ul -> 948 ng
- NegCtrl: 33.5 ng/ul -> 1,005 ng
- Exp: 39.3 ng/ul -> 1,179 ng
- With Qubit dsDNA HS
- PosCtrl: 1.44 ng/ul -> 43.2 ng
- NegCtrl: 1.15 ng/ul -> 34.5 ng
- Exp: 1.63 ng/ul -> 48.9 ng
- Nextera kit requires 50ng input DNA
- NexteraXT only requires 1ng
- The fact negative control has dsDNA is worrying
Gel Check[edit]
- 0.5ul Low Mass DNA Ladder + 2ul H2O + 2ul 6X Dye Buffer
- 2ul sample + 2ul 6X Dye Buffer