Notebook
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7/3
Restriction Enzyme Digestion
Tong, Tamukai
| component | volume(µl) |
|---|
| BSA | 0.5 |
| buffer(10xH) | 5 |
| EcoR1 | 0.5 |
| Pst1 | 0.5 |
| Dpn1 | 0.5 |
| dH2O | 18 |
add 4µL linearized plasmid backbone (25ng/µL)
add 4µL Enzyme Master Mix
digest 37℃, 30min
add 8µL NaOAc (pH5.2,3M)
add 16µL 99.5% EtOh
Vortex 2sec
add 184µL 99.5% EtOH
add 72µL DW
7/5
OE-PCR
Nambu, Shimazoe
| sample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | template DNA | volume (ul) | Q5 HF 2xMasterMix (ul) | DW (ul) |
|---|
| 1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 2 | Z008 | 1.25 | Z011 | 1.25 | GFP | 1.0 | 1.0 | 9.0 |
| 3 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 4 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
| 5 | Z013 | 1.25 | Z016 | 1.25 | RFP | 1.0 | 1.0 | 9.0 |
| 6 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 1.0 | 9.0 |
7/6
OE-PCR
Nambu, Shimazoe
| sample | Primer Forward | volume (ul) | Primer Reverse | volume (ul) | Template DNA | volume (ul) | Q5 HF2x Master Mix (ul) | DW (ul) |
|---|
| 1 | Z007 | 1.25 | Z009 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 2 | Z010 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 3 | Z007 | 1.25 | Z014 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 4 | Z015 | 1.25 | Z012 | 1.25 | Yeast genome | 1.0 | 12.5 | 9.0 |
| 5* | Z007 | 1.25 | Z009 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 6* | Z010 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 7* | Z007 | 1.25 | Z014 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
| 8* | Z015 | 1.25 | Z012 | 1.25 | p079 | 1.0 | 12.5 | 9.0 |
*sample5~8 x 2set
7/6
colony_PCR
| Mangrin colony | DW (ul) | Q5 HF2x MasterMix (ul) | m13 Forward (ul) | m13 Reverse (ul) |
|---|
| 1 | 2.0 | 2.5 | 0.25 | 0.25 |
| 2 | 2.0 | 2.5 | 0.25 | 0.25 |
| 3 | 2.0 | 2.5 | 0.25 | 0.25 |
| 4 | 2.0 | 2.5 | 0.25 | 0.25 |
| 5 | 2.0 | 2.5 | 0.25 | 0.25 |
Store PCR product at 4℃.
7/12
OE-PCR
Nambu
| sample | KOD one PCR master mix (ul) | TDH promoter | volume (ul) | ORF | volume (ul) | CYC terminator | volume (ul) | DW (ul) |
|---|
| A | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
| B | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |
| C | 12.5 | 5 | 1.5 | GFP | 1.5 | 6 | 1.5 | 8.0 |
| D | 12.5 | 7 | 1.5 | RFP | 1.5 | 8 | 1.5 | 8.0 |
PCR
98℃ 0:30
98℃* 0:10
45℃* 0:05
68℃* 0:05
68℃ 2:00
10℃
(* x 30 cycle)
