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

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
  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 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

File:2014-06-06 RTPrimerinvitroTest.jpg