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Team:DTU-Denmark-2/Team/Protocols
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===='''PCR Programs'''==== | ===='''PCR Programs'''==== | ||
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| + | !scope="col" | Temperature [°C] | ||
| + | !scope="col"| Time [min] | ||
| + | !scope="col"| Cycles | ||
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| + | !scope="row" align="center"| 98 | ||
| + | |align="center"| 2:00 | ||
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| + | !scope="row" align="center" | 98 | ||
| + | |align="center"|0:10 | ||
| + | |align="center"|35 | ||
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| + | !scope="row" align="center"| 59 | ||
| + | |align="center"| 0:30 | ||
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| + | !scope="row" align="center"| 72 | ||
| + | |align="center"| 3:00 | ||
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| + | !scope="row" align="center"| 72 | ||
| + | |align="center"| 5:00 | ||
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| + | !scope="row" align="center"| 12 | ||
| + | |align="center"| Store | ||
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===USER cloning=== | ===USER cloning=== | ||
Revision as of 08:25, 12 August 2011
Contents |
Protocols
Amplifying of biobricks by PCR
PCR MIX
| PCR mix | 1 x PCR mix á 50µl |
|---|---|
| 5 x HF PCR buffer with MgCl2 or GC buffer | 10µl |
| dNTP’s 2mM | 5µl |
| Primer forward 10 µM | 4µl |
| Primer reverse 10 µM | 4µl |
| Phusion DNA polymerase 5u/µl | 0.3µl |
| DNA template | 0.5µl |
| MilliQ water | 26.20µl |
PCR Programs
| Temperature [°C] | Time [min] | Cycles |
|---|---|---|
| 98 | 2:00 | |
| 98 | 0:10 | 35 |
| 59 | 0:30 | - |
| 72 | 3:00 | - |
| 72 | 5:00 | |
| 12 | Store |
USER cloning
Tranformation in E.coli
Purifying of plasmid
Fungi
Transformation in fungi
Genetic Transformation of filamentous fungi – protocol from Center for Microbial Biotechnology (CMB) at DTU, Author Nielsen, J. B.
Media
- Minimal medium (MM) (1L) -50 mL D-glucose 20% w/V, 20 mL 50x mineral mix, 10 mL 1 M sodium nitrate, 20 g ager.
- Transformation media(TM)(1L)- 342.3 g Sucrose, 20 mL 50x mineral mix, 20 g agar.
- Mineral Mix (1L)- 26g KCL, 26g MgSO4·7H2O, 76g KH2PO4, 50 mL Trace element solution, MIlli-Q water to volume 1000 mL
- D-glucose 20% (0.5 L) - 100g D-glucose and MilliQ water up to 500 ml
- Aspergillus protoplastationbuffer (APB)- Final conc. 1.1 M MgSO4 and 10 mM Na-phosphate buffer. pH is adjusted with 2 N NaOH to 5.8.
- Aspergillus transformation buffer (ATB)- Final conc: 1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.
- PCT (200ml) - Final conc: 50% w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store at 4 °C
Initiation: The host strain is grown as three-point stabs on Minimal medium plates with the require suppliants added. MM will for convenience throughout the protocol refer to MM with the supplements included.
Inoculation: The conidia are harvest by adding 5 ml of MM and firmly rub with a sterile Drigalsky spatula. The conidial suspension is pipette to a sterile 500 ml shake flask containing 100 ml MM. The cultures are incubated at 30 °C with 150 rpm of shaking over night (14-20 hours)
Mycelial harvest: A funnel with a sterile Mira cloth (filter) is used to harvest mycelia. To remove residual glucose from mycelia the biomass are wash with Aspergillus protoplastationbuffer (APB). The filtered biomass is transferred to a new Falcon tube with a sterile spoon.
Protoplastation: Mycellium is resuspenden in 10 ml filter-sterillized(0.45μm filters) APB containing 40 mg Glucanex/ml. The Glucanex is dissolved in APB with gentle magnetic stirring less than 100/min. Mycelia with dissolved Glucanex are mixed at 30 °C with 150 rpm of shaking for 2-3 hours.
Portoplast solutions are diluted in APB adding up to 40 ml mark. An overlay of max. 5ml Aspergillus transformation buffer (ATB) diluted to ½x with sterile MilliQ-water is carefully placed on top of the APB. Centrifuged at 13 min 3000 RCF in Sorvall centrifuge. Protoplates should be observed as a halo of with slurry in the interphase of the two liquids. Withdraw of the protoplasts are done with pipette and placed in a Falcon tube. ATB is added up to 40 ml mark. Centrifuge at 300 RCF in 13 min and supernatanted are discarded.
The protoplastes are resuspended in 1 ml ATB with a 5 ml pipette.
Genetic transformation: Aliquotes of 200 μl are transferred to a 1.5 ml Eppendorf tube containgen the DNA for transformation. DNA concentration with bipartite substrates should be 2x(1-5μl). Protoplast and DNA are incubated at room temperature for at least 30 min.
Protoplat and DNA suspension are added to 1 ml PCT in a 15 ml tube and shake gently. Incubated for 1-5 min at room temperature. Diluted in 3 ml ATB. The tube is filled with molten transformation medium (TM) agar (temperature of 40-45°C) to apporeimately 12 ml. The tube is mix rapidly by inverting the tube twice. Poured directly on pre-made TM plates and incubated at 37°C for 3-8 days.
