Matt:LabNotes/2016-11-10: Difference between revisions

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>Mzcai
>Mzcai
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===Qia Column Purification===
===Qia Column Purification===
*8 columns elute 50ul each<!--
*16 columns elute 50ul each
*Nanodrop: 208.8 ng/uL x 200uL = 41.76ug
*Nanodrop: 170.3 ng/uL x 800uL = 136.24ug


===Lambda Exo===
===Lambda Exo===
*Divide into 4 pcr tubes of 100ul (ideally should be <10ug each)
*Divide into 14 pcr tubes of 100ul (ideally should be <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||50
| Amplicon||57
|-
|-
| 10X Lambda Exo Buffer||10
| 10X Lambda Exo Buffer||10
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| Lambda Exonuclease||10
| Lambda Exonuclease||10
|-
|-
| H2O||30
| H2O||23
|-
|-
| 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 14 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each<!--
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**96.8ng/ul x 160ul = 15.49ug (74% yield)
**96.8ng/ul x 160ul = 15.49ug (74% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
*Save 4ul for gel check of size
====USER====
====USER====
*Split into 3 PCR tubes and add 5ul USER
*Split into 3 PCR tubes and add 5ul USER

Revision as of 02:49, 13 November 2016

CA12k_Oct2016 V4 Probe Preparation

Production PCR

  • V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Oct2016_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
  • Did one plate - 96 wells 100ul each

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold 450px

EtOH Precipitation

  • 16 5-ml tubes each with
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • 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 16 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

  • 16 columns elute 50ul each
  • Nanodrop: 170.3 ng/uL x 800uL = 136.24ug

Lambda Exo

  • Divide into 14 pcr tubes of 100ul (ideally should be <10ug each)
Components Volume
Amplicon 57
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 23
Total 100
  • Incubated at 37C for 1hr
  • Purified with 14 Zymo ssDNA/RNA columns
  • Eluted with 40ul each