Team:NTNU Trondheim/Protocols

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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 [http://products.invitrogen.com/ivgn/product/18265017 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 [http://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/0/wizard%20plus%20sv%20minipreps%20dna%20purification%20system%20protocol.pdf?la=en protocol] supplied by the vendor.

DNA Concentration measurements

Concentrations of DNA after isolation was measured with the [http://www.nanodrop.com/library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop ND-1000 Spectrophotometer].

Restriction digest

We are using the single reaction protocol from partsregistry.org : [http://partsregistry.org/Help:Protocols/Restriction_Digest]

Ligation

Ligation protocol from partsregistry.org [http://partsregistry.org/Help:Protocols/Ligation]:

Gel electrophoresis

Agarose with Gel Green is used when molding the gel. Choose an appropriate ladder as a referance to size of the fragments and put 2 µl of ladder in wells on either side of the samples. When applying the samples, add 20% loading dye to the sample. For instance, if the initial sample is 10 µl, add 2 µl loading dye. Apply the samples to individual wells in the gel.

Let the gel run for 45 min at 90 V, and if the bands are poorly separated, run a little longer. Beware that if the gel runs for a very long time, the smaller bands may move out of the gel.

Gel purification

We are using the QIAquick Gel Extraction kit, following the protocol from www.qiagen.com. [http://www.qiagen.com/literature/render.aspx?id=201083]

OD measurements

Unless otherwise noted, all OD measurements are made at 600 nm with a PerkinElmer Lambda 35 spectrometer, with un-inoculated medium as reference.

Fluorescence measurements

Fluorescence were measured with a Tecan Infinite M200 Pro microplate reader using Nunclon flat bottom black polystyrol 96 well plates.

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 uL glycerol solution and 800 uL 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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