Matt:LabNotes/2014-6-24: Difference between revisions

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==Removal of Amplificiation Adapters==
==Agi26k0gap Probe Production Continued==
 
===Removal of Amplification Adapters===
*[[Matt:LabNotes/2014-6-11#Lambda_Exonuclease_Digestion | Lambda digested 13.7ug ssDNA]]  
*[[Matt:LabNotes/2014-6-11#Lambda_Exonuclease_Digestion | Lambda digested 13.7ug ssDNA]]  
*[[Matt:LabNotes/2014-5-22#Lambda_Exonuclease_Digestion | Lambda digested 7.87ug ssDNA]]
*[[Matt:LabNotes/2014-5-22#Lambda_Exonuclease_Digestion | Lambda digested 7.87ug ssDNA]]
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Summary: 4 tubes of 200ul each
Summary: 4 tubes of 200ul each
===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

Revision as of 00:43, 26 June 2014

Agi26k0gap Probe Production Continued

Removal of Amplification Adapters

To each of 4 x 200ul PCR tubes:

Components Volume
ss-amplicon (73.3 ng/ul) 75
USER 5
H2O 0
total 80
 *5.5ug 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: 4 tubes of 200ul each

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