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

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*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**95.1 ng/ul x 160ul =  30.4 ug (46.5% yield)
**95.1 ng/ul x 320ul =  30.4 ug (46.5% yield)
***Maybe try less lambda digestion time? or more?
***Maybe try less lambda digestion time? or more?
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===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====
*Split into 4 PCR tubes and add 5ul USER
*Split into 6 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||59
| SS-amplicon||52
|-
|-
| USER||5
| USER||5
Line 82: Line 82:
| 10X DpnII Buffer||8
| 10X DpnII Buffer||8
|-
|-
| H2O||8
| H2O||15
|-
|-
| Total||80
| Total||80
Line 105: Line 105:
*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 20ul each column (80ul total)
*Eluted 20ul each column (80ul total)

Revision as of 23:07, 14 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)