Matt:LabNotes/2015-1-15: 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 50ul H2O
*Resuspended DNA with 50ul H2O
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===Qia Column Purification===
===Qia Column Purification===
*Purified in 6 columns following Qiagen protocol
*Purified in 6 columns following Qiagen protocol
Line 63: Line 63:
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:


~150ul of V4 probes: 127.8 ng/ul => ~37 ug
~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug
~150ul of V7 probes: 113.9 ng/ul => ~33 ug
~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide each into 4 pcr tubes of 72.5ul with total amplicon of <10ug each
*Divide each into 3 pcr tubes of 50ul 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||72.5
| Amplicon||50
|-
|-
| 10X Lambda Exo Buffer||10
| 10X Lambda Exo Buffer||10
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| Lambda Exonuclease||10
| Lambda Exonuclease||10
|-
|-
| H2O||7.5
| H2O||30
|-
|-
| Total||100
| Total||100
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*Incubated at 37C for 1hr
*Incubated at 37C for 1hr
*Purified with 4 Zymo ssDNA/RNA columns
*Purified with 3 Zymo ssDNA/RNA columns each
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**66.5ng/ul x 160ul = 10.6ug (57.3% yield)
**ng/ul x 120ul = ug (% yield)
**56.9ng/ul x 160ul = 9.1ug (55.2% yield)
**ng/ul x 120ul = ug (% yield)
 
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===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====

Revision as of 22:01, 18 January 2015

CA12k_Nov2014 V4 and V7 Probe Production

Production PCR

Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V4 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V4U (100uM) 0.4 20
AP2V4 (100uM) 0.4 20
H2O 49 2450
Total 100 5000
  • V7 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V7U (100uM) 0.4 20
AP2V7 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

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

EtOH Precipitation

  • 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
    • 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 50ul H2O

Qia Column Purification

  • Purified in 6 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another
  • Measured concentration with Nanodrop:

~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug

Lambda Exonuclease Digestion

  • Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 1hr
  • Purified with 3 Zymo ssDNA/RNA columns each
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • ng/ul x 120ul = ug (% yield)
    • ng/ul x 120ul = ug (% yield)