Matt:LabNotes/2015-2-2: 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 100ul H2O
*Resuspended DNA with 100ul H2O
<!--
 
===Qia Column Purification===
===Qia Column Purification===
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
Line 46: Line 46:
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:


~580ul of V4 probes: 93.2 ng/ul => ~27 ug
~575ul of V4 probes: 86.9 ng/ul => ~50 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
*Divide into 6 pcr tubes of ~100ul 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||100
| Amplicon||96
|-
|-
| 10X Lambda Exo Buffer||12.5
| 10X Lambda Exo Buffer||12
|-
|-
| Lambda Exonuclease||12.5
| Lambda Exonuclease||12
|-
|-
| Total||125
| Total||120
|}
|}


*Incubated at 37C for 1hr
*Incubated at 37C for 1hr
*Purified with 3 Zymo ssDNA/RNA columns
*Purified with 6 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each<!--
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**62.9ng/ul x 120ul = 7.5ug (56% yield)
**62.9ng/ul x 240ul = 7.5ug (56% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===

Revision as of 19:53, 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:020215 CA12kNov14 ProductionPCR V4.JPG

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

Qia Column Purification

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

~575ul of V4 probes: 86.9 ng/ul => ~50 ug

Lambda Exonuclease Digestion

  • Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
Components Volume
Amplicon 96
10X Lambda Exo Buffer 12
Lambda Exonuclease 12
Total 120
  • Incubated at 37C for 1hr
  • Purified with 6 Zymo ssDNA/RNA columns
  • Eluted with 40ul each