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Team:NTNU Trondheim/Protocols
From 2011.igem.org
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{{:Team:NTNU_Trondheim/NTNU_header|}} | {{:Team:NTNU_Trondheim/NTNU_header|}} | ||
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| + | =Protocols= | ||
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| + | ==Resuspending DNA from registry-parts:== | ||
| + | *Poke a hole in foil of corresponding well | ||
| + | *Resuspend DNA in 10 µL dH2O (pipette up and down a few times) | ||
| + | *Wait 5 minutes. | ||
| + | *Transfer the resuspended DNA to pCR tube and store in -20C. | ||
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| + | ==Transforming competent cells:== | ||
| + | *Thaw competent cells on ice | ||
| + | *Mix with 2 µL plasmid DNA | ||
| + | *Incubate on ice 30 minutes | ||
| + | *Heat-shock cells 45 seconds in 42C water-bath. | ||
| + | *Incubate 5 minutes on ice | ||
| + | *Add 200 µL SOC | ||
| + | *Incubate the Eppendorf tubes in Erlenmeyer flask 37C shaking incubator for 2 hours | ||
| + | *Plate 50 µl and 200 µl of the transformation onto the dishes, and spread. | ||
| + | *Incubate ON in 37C | ||
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= Recipes used in the lab = | = Recipes used in the lab = | ||
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Ampicillin 100 µg / mL (500 µL amp stock (200 mg/mL) in 500 mL agar) | Ampicillin 100 µg / mL (500 µL amp stock (200 mg/mL) in 500 mL agar) | ||
IPTG 0,1 mM ( 63,5 µL IPTG stock (0,8 M)) | IPTG 0,1 mM ( 63,5 µL IPTG stock (0,8 M)) | ||
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Revision as of 07:54, 5 July 2011
Protocols
Resuspending DNA from registry-parts:
- Poke a hole in foil of corresponding well
- Resuspend DNA in 10 µL dH2O (pipette up and down a few times)
- Wait 5 minutes.
- Transfer the resuspended DNA to pCR tube and store in -20C.
Transforming competent cells:
- Thaw competent cells on ice
- Mix with 2 µL plasmid DNA
- Incubate on ice 30 minutes
- Heat-shock cells 45 seconds in 42C water-bath.
- Incubate 5 minutes on ice
- Add 200 µL SOC
- Incubate the Eppendorf tubes in Erlenmeyer flask 37C shaking incubator for 2 hours
- Plate 50 µl and 200 µl of the transformation onto the dishes, and spread.
- Incubate ON in 37C
Recipes used in the lab
LB Broth
- Bacto-tryptone: 10 g/L
- Yeast extract: 5 g/L
- NaCl 5 g/L
Adjust pH to 7,4 with NaOH, autoclave.
LA
- Dissolve LB
- Add agar, 15 g/L
Adjust pH to 7,4 with NaOH, autoclave.
SOC:
- Bacto-tryptone: 20 g/L
- Yeast extract: 5 g/L
- NaCl 0,5 g/L
- 10 mL 250 mM KCl (25mM) → 0,186 g / 10mL H20
- 5 mL 2M MgCl2 (10mM) → 0,952 g / 5mL
Autoclave, cool, then add:
- 20 mL 1M sterile-filtered glucose (20mM)
Ampicillin-plates:
Use stock 200 mg/mL. → 100µg/mL in liquid agar. I.E. 250 µL stock in 500 mL agar. Add when cool enough to pour.
Spectinomycin-plates:
Use stock 100 mg/mL. → 100µg/mL in liquid agar. I.E. 500 µL stock in 500 mL agar. Add when cool enough to pour.
Ampicillin-IPTG plates:
Ampicillin 100 µg / mL (500 µL amp stock (200 mg/mL) in 500 mL agar) IPTG 0,1 mM ( 63,5 µL IPTG stock (0,8 M))
