Matt:LabNotes/2014-9-18: Difference between revisions

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>Mzcai
(Created page with "==Agi26k0gap Probe Production== ===Production PCR=== *Make Master Mix for 51 well (7 strips) *0 gap first round amplicon -> V6 primers {| {{table}} | align="center" style="b...")
 
>Mzcai
mNo edit summary
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98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold
98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold


[[650px]]
[[File:20140919_Agi26k0gapProductionPCR.PNG|650px]]


===EtOH Precipitation===
===EtOH Precipitation===
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**85ul 3M NaOAc pH 5.2-5.5
**85ul 3M NaOAc pH 5.2-5.5


*Vortexed and put in -80C for overnight<!--
*Vortexed and put in -80C for overnight
*Centrifuged at 3000rpm at 4C for 30min
*Centrifuged at 3000rpm at 4C for 30min
*Discarded supernatant and added 800ul of cold 80% EtOH
*Discarded supernatant and added 800ul of cold 80% EtOH
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===Qia Column Purification===
===Qia Column Purification===
*Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
*Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
*Eluted each column with 40ul and combined all probes into one 1.5ml
*Eluted each column with 40ul and combined all probes into one 1.5ml<!--
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:
*~225ul of Agi26k0gap probes: 317.5 ng/ul =>  71.4 ug
*~225ul of Agi26k0gap probes: 317.5 ng/ul =>  71.4 ug

Revision as of 21:03, 20 September 2014

Agi26k0gap Probe Production

Production PCR

  • Make Master Mix for 51 well (7 strips)
  • 0 gap first round amplicon -> V6 primers
Components 1x rxn (ul) 50x rxn (ul)
First round amplicon (10nM) 0.2 10
2x Kapa SYBR MM 50 2500
100uM pAP1V6U 0.4 20
100uM AP2V6 0.4 20
H2O 49 2450
Total 100 5000

98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold

File:20140919 Agi26k0gapProductionPCR.PNG

EtOH Precipitation

  • 6 5-ml tubes (with 8.5 wells of PCR product each) for 0gap
    • 850ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 85ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 3000rpm at 4C for 30min
  • 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 15,000rpm for 5 min on table-top centrifuge
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
  • Eluted each column with 40ul and combined all probes into one 1.5ml