Matt:LabNotes/2014-6-9

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Repeat Top48 Hexamer RT Primer in vitro Validation[edit]

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
  1. Heat reaction mix (above) to 65C for 5min
  2. Add 1ul 10X M-MuLV RT Buffer (total 10ul volume)
  3. Incubate 25C for 10min
  4. Add 1ul M-MuLV Reverse Transcriptase to Pos Ctrl and Exp ONLY (1ul H2O for Neg Ctrl)
  5. Incubate 42C for 60min
  6. Inactivate by heating to 85C for 10min

Second Strand Synthesis[edit]

  1. Add 57ul H2O
  2. Add 8ul 10X Second Strand Synthesis Buffer
  3. Add 4ul Second Strand Enzyme
  4. Mix by pipetting
  5. Incubate 16C for 2.5 hours
  6. 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

Continued on: Matt:LabNotes/2014-6-13

Qubit Quant[edit]

  • PosCtrl: 1.06ng/ul
  • NegCtrl:0.884ng/ul
  • Exp:0.818ng/ul