Matt:LabNotes/2014-6-24
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Agi26k0gap Probe Production Continued[edit]
Removal of Amplification Adapters[edit]
- Lambda digested 13.7ug ssDNA
- Lambda digested 7.87ug ssDNA
- Combine to 300ul of 22ug total ssDNA
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
- Continued on: Matt:LabNotes/2014-6-27