Matt:LabNotes/2014-9-25

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Revision as of 22:05, 25 September 2014 by >Mzcai (Created page with "==Repeat Top48 RT Primer in vitro Validation after DNA removal from UHRR== *Must first remove dsDNA from UHRR since there was contamination [[Matt:LabNotes/2014-6-18 | last ti...")
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Repeat Top48 RT Primer in vitro Validation after DNA removal from UHRR

  • Must first remove dsDNA from UHRR since there was contamination last time

Purify UHRR of DNA

  • Use Zymo DNA-Free RNA Kit
  • Sample: 4ul of UHRR (1.3 ug/ul)

DNase I Digest

Components Volume
UHRR (1.3ug/ul) 4
10X DNase I Buffer 5
DNase I 2
H2O 39
Total 50
  • Mix and incubate at 37C for 30min

Purify

  • Add 100ul RNA Binding Buffer and mix
  • Add 150ul 100% EtOH and mix
  • Transfer to Zymo-Spin IC Column in Collection Tube
    • Centrifuge at 13,000rcf for 1min and discard flow through
  • Add 400ul RNA Prep Buffer
    • Centrifuge at 13,000rcf for 1min and discard flow through
  • Add 800ul RNA Wash Buffer
    • Centrifuge at 13,000rcf for 30sec and discard flow through
  • Add 400ul RNA Wash Buffer
    • Centrifuge at 13,000rcf for 30sec and discard flow through
  • Centrifuge at 13,000rcf for 2min and discard flow through
  • Transfer column to 1.5ul tube and add 8ul H2O and let stand for 1min
    • Centrifuge at 10,000rcf for 30sec
  • Store at -20C