Matt:LabNotes/2017-4-12: 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 14 -> 72C 2min -> 15C hold
[[ | 800px]]
[[File:20170412_Agi15kFeb2017_ProductionPCR.png | 600px]]
*Do 12 or 13 cycles next time


===EtOH Precipitation===
===EtOH Precipitation===
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**120ul 3M NaOAc pH 5.2-5.5
**120ul 3M NaOAc pH 5.2-5.5


*Vortexed and put in -80C for overnight<!--
*Vortexed and put in -80C for overnight
*Centrifuged at 13000rpm at 4C for 20 min (in basement ultracentrifuge)
*Centrifuged at 10,000rpm at 4C for 30 min
*Discarded supernatant and added 800ul of cold 80% EtOH
*Discarded supernatant and added 800ul of cold 80% EtOH
*Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
*Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
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*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:
 
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~400ul of V4 probes: 111.1 ng/ul => ~44.4 ug
~400ul of V4 probes: 111.1 ng/ul => ~44.4 ug



Revision as of 18:02, 13 April 2017

Agi15k_Feb2017_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon Agi15k_Feb2017_V4 (25nM) 0.1 10
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4910
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold File:20170412 Agi15kFeb2017 ProductionPCR.png

  • Do 12 or 13 cycles next time

EtOH Precipitation

  • 8 5-ml tubes (with 12 wells of PCR product each) for V4
    • 1200ul PCR product
    • 3000ul 100% EtOH
    • 3ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 10,000rpm 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 15 min at 4C
  • Discarded supernatant and air-dried for 10 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop: