Team:NTNU Trondheim/Protocols

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Protocols

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

Contents

1 Transformation
2 Inoculation after transformation
3 DNA Isolation
4 DNA Concentration measurements
5 Restriction digest
6 Ligation
7 Gel purification
8 Recipes
- 8.1 Growth media
- 8.2 Antibiotics

Transformation

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

We use this protocol with the following modifications:

45 s heat shock in stead of 60 s.
LB medium in stead of SOC.

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]

250 ng of DNA is added together with the appropriate amount of dH2O, for a total volume of 16 uL.
Add 2,5 uL of NED-buffer 2
Add 0,5 uL og BSA
Add 0,5 uL of Enzyme 1
Add 0,5 uL of Enzyme 2
The total volume should now be 20 uL. Mix well and spin down.
Incubate the restriction digest at 37C for 1h
Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.

Ligation

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

Add 11 uL of dH2O
Add 2 uL of each sample to be ligated (insert and backbone)
Add 2ul of T4 DNA Ligase Reaction Buffer
Add 1ul of T4 DNA Ligase
Mix well, and spin down
Incubate for 30min at 16C and 20min at 80C to heat kill
Use 2ul of ligation to transform into competent cells

Gel purification

Uses the QIAquick PCR Purification kit, following the protocol: [http://www.qiagen.com/literature/render.aspx?id=201083]

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.

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