Matt:LabNotes/2014-7-22: Difference between revisions
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>Mzcai (Created page with "==Agi26k0gap Probe Production Continued== ===Removal of Amplification Adapters=== *[[Matt:LabNotes/2014-7-1#Lambda_Exonuclease_Digestion | Lambda digested 14.2ug (200ul) ssDNA...") |
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*[[Matt:LabNotes/2014-7-1#Lambda_Exonuclease_Digestion | Lambda digested 14.2ug (200ul) ssDNA]] | *[[Matt:LabNotes/2014-7-1#Lambda_Exonuclease_Digestion | Lambda digested 14.2ug (200ul) ssDNA]] | ||
*[[Matt:LabNotes/2014-7-17#Lambda_Exonuclease_Digestion | Lambda digested 21.4ug (320ul)ssDNA]] | *[[Matt:LabNotes/2014-7-17#Lambda_Exonuclease_Digestion | Lambda digested 21.4ug (320ul)ssDNA]] | ||
*Combine to | *Combine to 520ul of 35ug total ssDNA | ||
To each of 7 x 200ul PCR tubes: | To each of 7 x 200ul PCR tubes: | ||
Line 10: | Line 10: | ||
| align="center" style="background:#f0f0f0;"|'''Volume''' | | align="center" style="background:#f0f0f0;"|'''Volume''' | ||
|- | |- | ||
| ss-amplicon | | ss-amplicon||71.5 | ||
|- | |- | ||
| USER||5 | | USER||5 | ||
|- | |- | ||
| H2O|| | | H2O||3.5 | ||
|- | |- | ||
| total||80 | | total||80 | ||
Line 51: | Line 51: | ||
Summary: 7 tubes of 200ul each | Summary: 7 tubes of 200ul each | ||
*Continued on: [[Matt:LabNotes/2014-8-4]] | |||
* | |||
[[ | |||
Latest revision as of 18:26, 4 August 2014
Agi26k0gap Probe Production Continued[edit]
Removal of Amplification Adapters[edit]
- Lambda digested 14.2ug (200ul) ssDNA
- Lambda digested 21.4ug (320ul)ssDNA
- Combine to 520ul of 35ug total ssDNA
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
- Continued on: Matt:LabNotes/2014-8-4