Matt:LabNotes/2015-5-5: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
Line 39: Line 39:
*Discarded supernatant and air-dried for 30 min in hood
*Discarded supernatant and air-dried for 30 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 45: Line 45:
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:


~600ul of V4 probes: ng/ul => ~ ug
~600ul of V4 probes: 59.8 ng/ul => ~35.9 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
*Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
**One of the 6 tubes somehow lost ~40% volume (I think when I was tapping the tubes it splashed out)
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
Line 66: Line 65:
*Purified with 6 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:<!--
**66.6 ng/ul x 240ul =  15.98 ug( 65% yield)
**66.6 ng/ul x 240ul =  15.98 ug( 65% yield)



Revision as of 18:48, 6 May 2015

CA12k_Nov2014_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V4 (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:20150505 CA12kNov2014 V4 ProductionPCR.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 12 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 30 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:

~600ul of V4 probes: 59.8 ng/ul => ~35.9 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
  • Recombined and measured ssDNA with Nanodrop: