Matt:LabNotes/2014-7-22: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| ss-amplicon (73.3 ng/ul)||75
| ss-amplicon||71.5
|-
|-
| USER||5
| USER||5
|-
|-
| H2O||0
| H2O||3.5
|-
|-
| total||80
| total||80
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Summary: 7 tubes of 200ul each
Summary: 7 tubes of 200ul each


<!--
*Continued on: [[Matt:LabNotes/2014-8-4]]
===TBU Gel Check===
*2ul Sample + 2ul 2X Urea Loading Dye
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
*Undigested sample: 10nM 1st round amplicon
 
[[File:2014-06-25_Agi26k0gapDigestCheck.jpg]]
*Brightest band below 200bp as expected
 
===Column Purification===
*Used Zymo ssDNA/RNA columns
*Total Eluted: 90ul
 
*Nanodrop quant: 142.9 ng/ul
*Yield: 142.9 ng/ul x 90 ul = 12.861 ug -->

Latest revision as of 18:26, 4 August 2014

Agi26k0gap Probe Production Continued[edit]

Removal of Amplification Adapters[edit]

To each of 7 x 200ul PCR tubes:

Components Volume
ss-amplicon 71.5
USER 5
H2O 3.5
total 80
 *5ug ssDNA per tube
  • Incubated at 37C for 2 hours
  • Added 15ul of the following
Components Volume
10X DpnII Buffer 10
100uM RE-DpnII guide oligo 5
 RE-DpnII guide oligo:
 0-gap: RE DpnII V6
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for 3 hr
  • Added 100ul of the following
Components Volume
10X DpnII Buffer 10
USER 5
H2O 85
Total 100
  • Incubated at 37C for overnight (~15 hrs)

Summary: 7 tubes of 200ul each