Matt:LabNotes/2014-6-9

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Revision as of 23:14, 9 June 2014 by >Mzcai (Created page with "==Repeat Top48 Hexamer RT Primer in vitro Validation== * Last time negative control ===Procedure=== ==...")
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Repeat Top48 Hexamer RT Primer in vitro Validation

Procedure

First Strand Synthesis

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

  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