Matt:LabNotes/2015-2-2: Difference between revisions

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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
[[File: | 800px]]
[[File: | 800px]]
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===EtOH Precipitation===
===EtOH Precipitation===
*12 5-ml tubes (with 8 wells of PCR product each) for V4
*12 5-ml tubes (with 8 wells of PCR product each) for V4
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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===
*Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
*Eluted each column with 50ul and combined all into one 1.5ml
*Eluted each column with 50ul and combined all into one 1.5ml
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:


~290ul of V7 probes: 93.2 ng/ul => ~27 ug
~580ul of V4 probes: 93.2 ng/ul => ~27 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===

Revision as of 00:44, 3 February 2015

CA12k_Nov2014_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V7 (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 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]

EtOH Precipitation

  • 12 5-ml tubes (with 8 wells of PCR product each) for V4
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 30min
  • 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 6 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