Matt:LabNotes/2015-1-13: Difference between revisions

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**80ul 3M NaOAc pH 5.2-5.5
**80ul 3M NaOAc pH 5.2-5.5


*Vortexed and put in -80C for overnight<!--
*Vortexed and put in -80C for overnight
*Centrifuged at 3000rpm at 4C for 30 min
*Centrifuged at 3000rpm at 4C for 30 min
*Discarded supernatant and added 800ul of cold 80% EtOH
*Discarded supernatant and added 800ul of cold 80% EtOH
*Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
*Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
*Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
*Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
*Discarded supernatant and air-dried for 5 min in hood
*Discarded supernatant and air-dried for 5 min in hood
Line 41: Line 41:


===Qia Column Purification===
===Qia Column Purification===
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
*Purified 6 tubes of 100ul 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
*Eluted each column with 50ul and combined all into one 1.5ml
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:


~290ul of V4 probes: 127.8 ng/ul => ~37 ug
~290ul of V7 probes: 93.2 ng/ul => ~27 ug
~290ul of V7 probes: 113.9 ng/ul => ~33 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 ~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||72.5
| Amplicon||100
|-
|-
| 10X Lambda Exo Buffer||10
| 10X Lambda Exo Buffer||12.5
|-
|-
| Lambda Exonuclease||10
| Lambda Exonuclease||12.5
|-
|-
| H2O||7.5
| Total||125
|-
| Total||100
|}
|}


*Incubated at 37C for 1hr
*Incubated at 37C for 1hr
*Purified with 4 Zymo ssDNA/RNA columns
*Purified with 3 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**66.5ng/ul x 160ul = 10.6ug (57.3% yield)
**62.9ng/ul x 120ul = 7.5ug (56% yield)
**56.9ng/ul x 160ul = 9.1ug (55.2% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====
*Split each set into 2 PCR tubes and add 5ul USER
*Save 2ul for gel check later
*Split into 2 PCR tubes and add 5ul USER
{| {{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'''
|-
|-
| SS-amplicon||80
| SS-amplicon||59
|-
|-
| USER||5
| USER||5
|-
|-
| Total||85
| 10X DpnII Buffer||8
|-
| H2O||8
|-
| Total||80
|}
|}
*Incubate at 37C for 2.5 hours
*Incubate at 37C for 1.5 hours
====DpnII====
====DpnII====
*Added 15ul of the following
*Added 15ul of the following
Line 92: Line 93:
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| 10X DpnII Buffer||10
| 10X DpnII Buffer||2
|-
| 100uM RE-DpnII_V7 guide oligo||5
|-
| H2O||8
|-
|-
| 100uM RE-DpnII guide oligo||5
|}
|}
  RE-DpnII guide oligo:
 
  V4: RE DpnII V4
  V7: RE DpnII V7
*Incubated at 94C for 2 min, then 37C for 3 min
*Incubated at 94C for 2 min, then 37C for 3 min
*Added 5ul DpnII
*Added 5ul DpnII
*Incubated at 37C for 4 hr
*Incubated at 37C for overnight
 
<!--
===TBU Gel: Digestion Check===
===TBU Gel: Digestion Check===
*Combine into 1 tube for each set
*Combine into 1 tube for each set

Revision as of 01:31, 14 January 2015

CA12k_Nov2014_V7 Probe Production

Production PCR

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 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:011214 CA12kNov14 ProductionPCR V7.JPG

EtOH Precipitation

  • 6 15-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 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 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 6 tubes of 100ul in 6 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~290ul of V7 probes: 93.2 ng/ul => ~27 ug

Lambda Exonuclease Digestion

  • Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
Components Volume
Amplicon 100
10X Lambda Exo Buffer 12.5
Lambda Exonuclease 12.5
Total 125
  • Incubated at 37C for 1hr
  • Purified with 3 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 62.9ng/ul x 120ul = 7.5ug (56% yield)

Remove Amplification Adapters

USER

  • Save 2ul for gel check later
  • Split into 2 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 1.5 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