Matt:LabNotes/2015-6-3: Difference between revisions

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*Total volume: 300ul
*Total volume: 300ul
*Nanodrop quantification: 155.7 ng/ul -> 46.7 ug
*Nanodrop quantification: 155.7 ng/ul -> 46.7 ug
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===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===
===Lambda Exonuclease Digestion===
*Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
*Divide into 6 pcr tubes of 50ul 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||96
| Amplicon||50
|-
|-
| 10X Lambda Exo Buffer||12
| 10X Lambda Exo Buffer||10
|-
|-
| Lambda Exonuclease||12
| Lambda Exonuclease||10
|-
|-
| Total||120
| H2O||30
|-
| Total||100
|}
|}


Line 89: Line 69:
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**79.2ng/ul x 240ul = 19ug (76% yield)
**ng/ul x 240ul = ug (76% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
Line 127: Line 107:
*Added 5ul DpnII
*Added 5ul DpnII
*Incubated at 37C for overnight (~15hrs)
*Incubated at 37C for overnight (~15hrs)
 
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===Zymo Column Purification===
===Zymo Column Purification===
*Eluted 30ul each column (115ul total)
*Eluted 30ul each column (115ul total)

Revision as of 19:47, 4 June 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

QIAEX-II Purification

protocol

  • Divide into 8 5mL tubes of 1200ul each
  • Add 3600ul (3X volume) of Buffer QX1
  • Vortex the QIAEXII for 30sec
  • Add 30ul QIAEXII (30ul for every 10ug)
  • Incubate at 50C while mixing for 10min
    • Color should be yellow
  • Centrifuge sample for 30sec
  • Remove supernatant with a pipette
  • Wash the pellet with 500ul Buffer QX1
  • Resuspend the pellet by vortexing
  • Transfer to 1.5ml tubes
  • Centrifuge sample for 30sec and remove supernatant again with pipette
  • Wash the pellet twice with 500ul Buffer PE
  • Air-dry the pellet for 30min until the pellet turns white
  • To elute, resuspend with 20ul H2O and vortexing
  • Incubate at RT for 5min
  • Centrifuge for 30sec and pipette out supernatant with sample
  • Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
  • Total volume: 300ul
  • Nanodrop quantification: 155.7 ng/ul -> 46.7 ug

Lambda Exonuclease Digestion

  • Divide into 6 pcr tubes of 50ul with total amplicon of <10ug each
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 1hr
  • Purified with 6 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • ng/ul x 240ul = ug (76% 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_V4 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)