Matt:LabNotes/2014-6-24

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Agi26k0gap Probe Production Continued[edit]

Removal of Amplification Adapters[edit]

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[edit]

  • 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[edit]

  • 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