Matt:LabNotes/2015-2-5: Difference between revisions

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>Mzcai
>Mzcai
Line 109: Line 109:
*Eluted 30ul each column
*Eluted 30ul each column
*Nanodrop
*Nanodrop
**46.6ng/ul x 120ul = 5.6ug (% yield)
**46.6ng/ul x 120ul = 5.6ug (30% yield)
<!--
 
===PAGE Size Selection===
===PAGE Size Selection===
*Run 3 gels
*Run 2 gels
**200V for 40min
**200V for 40min
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''3X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| V4 Probes||115
| V7 Probes||120
|-
|-
| TBE-Urea Buffer 2X||115
| TBE-Urea Buffer 2X||120
|}
|}


{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''3X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| Low Mass Ladder||3
| Low Mass Ladder||2
|-
|-
| TBE-Urea Buffer 2X||15
| TBE-Urea Buffer 2X||10
|-
|-
| H2O||12
| H2O||8
|-
|-
| Total||30
| Total||20
|}
|}


*Gel 1
*Gel 1
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel1.jpg]]
 
*Gel 2
*Gel 2
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel2.jpg]]
 
*Gel 3
<!--
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel3.jpg]]
===EtOH Precipitation===
===EtOH Precipitation===
*Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.

Revision as of 22:10, 9 February 2015

CA12k_Nov2014_V7 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
AP1V7U (100uM) 0.4 40
AP2V7 (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: | 800px]]

EtOH Precipitation

  • 12 5-ml tubes (with 8 wells of PCR product each) 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 90min
  • 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:

~590ul of V7 probes: 107.3ng/ul => 63.3ug

Lambda Exonuclease Digestion

  • Divide into 6 pcr tubes of ~100ul with total amplicon of ~10ug each
Components Volume
Amplicon 98
10X Lambda Exo Buffer 12
Lambda Exonuclease 12
Total 122
  • Incubated at 37C for 1hr
  • Purified with 6 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 78.1ng/ul x 240ul = 18.7ug (59% yield)

Remove Amplification Adapters

USER

  • Split into 4 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 59
USER 5
10X DpnII Buffer 8
H2O 8
Total 80
  • Incubate at 37C for 2.5 hours

DpnII

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 2
100uM RE-DpnII_V7 guide oligo 5
H2O 8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (~15hrs)

Zymo Column Purification

  • Eluted 30ul each column
  • Nanodrop
    • 46.6ng/ul x 120ul = 5.6ug (30% yield)

PAGE Size Selection

  • Run 2 gels
    • 200V for 40min
Components 2X Volume
V7 Probes 120
TBE-Urea Buffer 2X 120
Components 2X Volume
Low Mass Ladder 2
TBE-Urea Buffer 2X 10
H2O 8
Total 20
  • Gel 1
  • Gel 2