Team:NTNU Trondheim/Protocols

From 2012.igem.org

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When transforming low copy number plasmids, it may be necessary to reduce the antibiotic to allow for the reduced antibiotic resistance of the tranformant.
When transforming low copy number plasmids, it may be necessary to reduce the antibiotic to allow for the reduced antibiotic resistance of the tranformant.
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===Glycerol stocks===
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Prepare a solution of 60 % glycerol in water. Add 400 mL glycerol solution and 800 mL of the culture to be stored in a cryogenic tube. Place in 5 C refrigerator for 30 min, then move to -80 C freezer.
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Revision as of 08:26, 13 July 2012

NTNU IS B.A.C.K.
Bacterial Anti-Cancer-Kamikaze

Protocols

This is a list of recipes and protocols used by the team.

Contents

Transformation

Official iGEM transformation protocol: http://partsregistry.org/Help:Protocols/Transformation

We use this protocol with the following modifications:

The cells used are Subcloning Efficiency™ DH5α™ Competent Cells from Life Technologies/Invitrogen

Inoculation after transformation

Using a sterile toothpick, pick/scratch a single colony from the transformants. Drop the toothpick into a plastic tube with 3 mL sterile liquid medium with the appropriate antibiotic(s). Close the tube but leave the cap slightly open to allow oxygen to enter, and incuate at 37 C with shaking.

DNA Isolation

We use the Promega Wizard Plus SV Minipreps DNA Purification System A1460 with the sentrifugation version of the protocol supplied by the vendor.

DNA Concentration measurements

Concentrations of DNA after isolation was measured with the NanoDrop ND-1000 Spectrophotometer.

Restriction digest

We are using the single reaction protocol from partsregistry.org : [1]

Ligation

Ligation protocol from partsregistry.org [2]:

In stead of incubating as described above, the samples are kept at 37 degrees celsius in a water bath for 60 minutes.

Gel purification

We are using the QIAquick Gel Extraction kit, following the protocol from www.qiagen.com. [3]

Recipes

Growth media

LB-medium (LB-Lennox):

Antibiotics

Ampicillin stock solutions: 100 mg/mL dissolved in MQ water
Kanamycin stock solutions: 100 mg/mL dissolved in MQ water
Chloramphenicol: 34 mg/mL dissolved in 100% ethanol

Store at -20 C after preparation and between use.

Ampicillin media concentration: 100 ug/mL = 1 mL stock solution/L medium
Kanamycin media concentration: 100 ug/mL = 1 mL stock solution/L medium Chloramphenicol media concentration: 24 ug/mL = 0.7 mL stock solution/L medium

When transforming low copy number plasmids, it may be necessary to reduce the antibiotic to allow for the reduced antibiotic resistance of the tranformant.

Glycerol stocks

Prepare a solution of 60 % glycerol in water. Add 400 mL glycerol solution and 800 mL of the culture to be stored in a cryogenic tube. Place in 5 C refrigerator for 30 min, then move to -80 C freezer. </div>

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