Matt:LabNotes/2017-4-12: Difference between revisions

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*Added 5ul DpnII
*Added 5ul DpnII
*Incubated at 37C for overnight (~15hrs)
*Incubated at 37C for overnight (~15hrs)
<!--
 
===Zymo Column Purification===
===Zymo Column Purification===
*Eluted 20ul each column (80ul total)
*Eluted 20ul each column (80ul total)
*Nanodrop
*Nanodrop
**103.7ng/ul x 80ul = 8.3ug (56% yield)
**114.9ng/ul x 120ul = 13.8ug (% yield)


===PAGE Size Selection===
===PAGE Size Selection===
*Run 2 gels
*Run 4 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;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
|-
|-
| V4 Probes||80
| V4 Probes||120
|-
|-
| TBE-Urea Buffer 2X||80
| TBE-Urea Buffer 2X||120
|}
|}


{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
|-
|-
| Low Mass Ladder||2
| Low Mass Ladder||4
|-
|-
| TBE-Urea Buffer 2X||10
| TBE-Urea Buffer 2X||20
|-
|-
| H2O||8
| H2O||16
|-
|-
| Total||20
| Total||40
|}
|}
[[File:2015-05-13_CA12kNov2014_V4_SizeSelect.jpg|450px]]
[[.jpg|450px]]


===EtOH Precipitation===
===EtOH Precipitation===
*Put cut out gel in 4 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Put cut out gel in 8 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
*Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
*Added 450 ul of 1X TE buffer to each
*Added 450 ul of 1X TE buffer to each
*Vortexed for 60min at 37 C in incubator
*Vortexed for 60min at 37 C in incubator<!--
*Centrifuged at 15,000 rpm for 3 min at RT
*Centrifuged at 15,000 rpm for 3 min at RT
*Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
*Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min

Revision as of 20:15, 17 April 2017

Agi15k_Feb2017_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon Agi15k_Feb2017_V4 (25nM) 0.1 10
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4910
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
File:20170412 Agi15kFeb2017 ProductionPCR.png

  • Do 12 or 13 cycles next time

EtOH Precipitation

  • 8 5-ml tubes (with 12 wells of PCR product each) for V4
    • 1200ul PCR product
    • 3000ul 100% EtOH
    • 3ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 10,000rpm at 4C for 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 15 min at 4C
  • Discarded supernatant and air-dried for 10 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~400ul of V4 probes: 327.7 ng/ul => ~131 ug

Lambda Exonuclease Digestion

  • Divide into 4 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 2hr
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 95.1 ng/ul x 320ul = 30.4 ug (46.5% yield)
      • Maybe try less lambda digestion time? or more?

Remove Amplification Adapters

USER

  • Split into 6 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 52
USER 5
10X DpnII Buffer 8
H2O 15
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)

Zymo Column Purification

  • Eluted 20ul each column (80ul total)
  • Nanodrop
    • 114.9ng/ul x 120ul = 13.8ug (% yield)

PAGE Size Selection

  • Run 4 gels
    • 200V for 40min
Components 4X Volume
V4 Probes 120
TBE-Urea Buffer 2X 120
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40

450px

EtOH Precipitation

  • Put cut out gel in 8 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
  • Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
  • Added 450 ul of 1X TE buffer to each
  • Vortexed for 60min at 37 C in incubator