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Team:Stockholm/1 September 2010
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Andreas
His⋅SOD cloning into pMA
Digestion
[pSB1C3.m-SOD]=105.5 ng/μl
[pMA.His]=85.67 ng/μl
| [μl] | SOD | pMA.His |
|---|---|---|
| 10X FD buffer | 3 | 3 |
| DNA (2 μg) | 19 | 23 |
| dH2O | 6 | 3 |
| FD AgeI | 0 | 0.5 |
| NgoMIV | 1 | 0 |
| FD SpeI | 1 | 1 |
| 30 | 30.5 |
Incubation:
- 37 °C, 0:30 (FD)
- 37 °C, 2:00 (NgoMIV)
Inactivation:
- 80 °C, 10 min
Ligation
| [μl] | |
| 5X Rapid ligation buf. | 4 |
| Vector DNA (pMa.His) | 1.5 |
| Insert DNA (m-SOD) | 4.5 |
| dH2O | 9 |
| T4 DNA ligase | 1 |
| 20 |
|---|
Incubation:
- 22 °C, 10 min
Transformation
Quick-transformation according to protocol into both Top10 and DH5α to compare and verify Top10 competence:
- 1.5 μl
- Amp 100
SOD⋅His cloning into pMA
Glycerol stocks
From 31/8 ON cultures
- SH1: pMA.SOD⋅His 1, 1/9/2010
- SH2: pMA.SOD⋅His 2, 1/9/2010
Transfer of m-yCCS into pEX
Digestion
[pEX.RFP] = 82 ng/μl [pSB1C3.m-yCCS] = 101.2 ng/μl
| [μl] | SOD | pMA.His |
|---|---|---|
| 10X FD buffer | 3 | 3 |
| DNA (1 μg) | 12 | 6 |
| dH2O | 13 | 19 |
| FD XbaI | 1 | 1 |
| FD PstI | 1 | 1 |
| 30 | 30 |
Incubation:
- 37 °C, 10 min
Inactivation:
- 65 °C, 20 min
Ligation
| [μl] | |
| 5X Rapid ligation buf. | 4 |
| Vector DNA (pEX) | 1.5 |
| Insert DNA (m-yCCS) | 9 |
| dH2O | 4.5 |
| T4 DNA ligase | 1 |
| 20 |
|---|
Incubation:
- 22 °C, 10 min
Transformation
Quick-transformation (Top10)
- 2 μl
- Amp 100
Mimmi
MITF-M, starting over...
Colony PCR
| Mix | (µl) | Primers | Conditions | ||||
|---|---|---|---|---|---|---|---|
| sH2O | 22.5 | MITF_FB_F_18aug | time | °C | |||
| F primer | 1 | MITF_FB_R_18aug | 2m | 95 | |||
| R Primer | 1 | 30s | 95 | ) | |||
| DNA | 0.5 | 30s | 55 | > 5 cycles | |||
| tot | 25µl | 1m40s | 72 | ) | |||
| 30s | 95 | \ 25 cycles | |||||
| 2m10s | 72 | / | |||||
| 10m | 72 | ||||||
| oo | 10 | ||||||
Gel
| well | sample |
|---|---|
| 1 | 1kb ladder |
| 2 | MITF-M + FB |
Digestion
| Mix | (µl) | (µl) | Conditions | ||
|---|---|---|---|---|---|
| MITF/pSB1C3 | 22 | 20 | Time | °C | |
| s2O | 10 | 5 | 30m | 37 | |
| 10x buffer | 4 | 3 | 20m | 65 | |
| EcoRI | 2 | 1 | oo | 10 | |
| SpeI | 2 | 1 | |||
| tot | 40µl | 30µl | |||
Ligation
| Mix | (µl) | Conditions | ||
|---|---|---|---|---|
| pSB1C3 | 3 | Time | °C | |
| MITF-M | 10 | 10m | 22 | |
| 5x buffer | 4 | oo | 4 | |
| T4 ligase | 1 | |||
| s2O | 2 | |||
| tot | 20µl | |||
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