Matt:LabNotes/2016-9-14: Difference between revisions

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*Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
*Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:
<!--
~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug
~600ul of V6 probes: 158.9 ng/ul => ~ 46 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide each into 6 pcr tubes of 48ul with total amplicon of <10ug each
*Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| Amplicon||48
| Amplicon||75
|-
|-
| 10X Lambda Exo Buffer||10
| 10X Lambda Exo Buffer||10
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| Lambda Exonuclease||10
| Lambda Exonuclease||10
|-
|-
| H2O||32
| H2O||5
|-
|-
| Total||100
| Total||100
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*Purified with 12 Zymo ssDNA/RNA columns
*Purified with 12 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:<!--
**V6: 64.0ng/ul x 240ul = 15.4ug (67% yield)
**V6: 64.0ng/ul x 240ul = 15.4ug (67% yield)



Revision as of 22:02, 16 September 2016

CA12k_Nov2014 V6 (RevComp of V4) Probe Production

Production PCR

  • V6 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V6 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V6U (100uM) 0.4 20
AP2V6 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold

EtOH Precipitation

  • 12 5-ml tubes (with 8 wells of PCR product each)
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 1hr
  • 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 12 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

  • Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
  • Measured concentration with Nanodrop:

~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug

Lambda Exonuclease Digestion

  • Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each
Components Volume
Amplicon 75
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 5
Total 100
  • Incubated at 37C for 1hr
  • Purified with 12 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop: