Matt:LabNotes/2014-6-9
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Repeat Top48 Hexamer RT Primer in vitro Validation[edit]
- Last time negative control
Procedure[edit]
First Strand Synthesis[edit]
Component | Pos Ctrl | Neg Ctrl | Exp |
25mM dNTP | 1 | 1 | 1 |
1.3ug/ul UHRR | 0.5 | 0.5 | 0.5 |
100uM RanHex Primer | 0.5 | 0.5 | 0 |
100uM Top48 Primer | 0 | 0 | 0.5 |
H2O | 7 | 7 | 7 |
- 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 magnetic bead (120ul beads to 80ul reaction)
- Elute with 25ul H2O
Gel Check[edit]
- 2ul sample + 2ul 6X loading dye
- 0.5ul Low Mass Lader + 2ul H2O + 2ul 6X loading dye
- First 2 gels ripped because stuck to the walls of the cassette
File:2014-06-10 RTPrimerinvitroTest.jpg
- Positive and negative control look the same
- Talking to Andrew, he pointed out that gels can't differentiate between single stranded, double stranded, and DNA vs RNA
- Possibly continue with Nextera since that will definitely tell us whether it worked or not
- Talking to Andrew, he pointed out that gels can't differentiate between single stranded, double stranded, and DNA vs RNA
Continued on: Matt:LabNotes/2014-6-13
Qubit Quant[edit]
- PosCtrl: 1.06ng/ul
- NegCtrl:0.884ng/ul
- Exp:0.818ng/ul