Matt:LabNotes/2016-7-26: Difference between revisions

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*Discarded supernatant and air-dried for 5 min in hood
*Discarded supernatant and air-dried for 5 min in hood
*Resuspended DNA with 100ul H2O
*Resuspended DNA with 100ul H2O
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===Qia Column Purification===
===Qia Column Purification===
*8 columns elute 50ul each
*8 columns elute 50ul each
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*Incubated at 37C for 2hr (normally 1hr)
*Incubated at 37C for 2hr (normally 1hr)<!--
*Purified with 8 Zymo ssDNA/RNA columns
*Purified with 8 Zymo ssDNA/RNA columns
**Dropped one column so only 7
**Dropped one column so only 7

Revision as of 18:47, 27 July 2016

TB12k_Apr2016 V4 Probe Preparation

Production PCR

  • Make 10ml V4 master mix for 96 wells
Components Volume (1X) Volume (100X)
First round amplicon TB12k_Apr2016_V4 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4900
Total 100 10,000

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold

  • Did in regular thermocyclers

EtOH Precipitation

  • 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 30 min
  • Centrifuged at 3000rpm at 4C for 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • 8 columns elute 50ul each
  • Nanodrop: 313.6 ng/uL x 400uL = 125.44ug

Lambda Exo

  • Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 2hr (normally 1hr)