Contents 1 Laboratory procedures 1.1 Transformation 1.2 Inoculation after transformation 1.3 DNA Isolation 1.4 DNA Concentration measurements 1.5 Restriction digest 1.6 Ligation 1.7 Linearized plasmids 1.8 Gel electrophoresis 1.9 Gel purification 1.10 Preparation of samples for RNA isolation 1.11 RNA isolation 1.12 DNAse reaction 1.13 cDNA reaction 1.14 OD measurements 1.15 Fluorescence measurements 2 Recipes 2.1 Growth media 2.2 Antibiotics 2.3 Glycerol stocks 2.4 Cake

Laboratory procedures

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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 the appropriate NEBuffer
• 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

Remember to do religation of backbone!!!!!!!!

Linearized plasmids

[http://partsregistry.org/Help:Protocols/Linearized_Plasmid_Backbones Official iGEM protocol]

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]

Preparation of samples for RNA isolation

• Grow overnight cultures
• Mix 1 ml cell culture with 2 ml RNA-protect (QIAGEN)
• Vortex for 5 seconds
• Incubate for 5 minutes in room temperature
• Spin down at 6000 g for 10 minutes in 4 °C

RNA isolation

(RNAquenous kit from Ambion)

• Add 100 µl Lysozyme/TE-mix to each sample
• Incubate for 5 minutes in room temperature
• Add 300 µl lysis/binding solution
• Vortex to make sure everything is solved
• Add 400 µl water for 64 % ethanol
• Turn the tubes 4 times, and transfer to filtertubes
• Centrifuge for 1 minute at 13000 g
• Add 700 µl wash solution 1
• Centrifuge for 1 minute at 13000 g
• Add 500 µl wash solution 2/3
• Centrifuge for 1 minute at 13000 g
• Add 500 µl wash solution 2/3
• Centrifuge for 1 minute at 13000 g
• Centrifuge for an additional minute at 13000 g
• Add 40 µl preheated elution buffer
• Centrifuge for 1 minute at 13000 g
• Add 20 µl preheated elution buffer
• Centrifuge for 1 minute at 13000 g
• Measure concentrations on NanoDrop

DNAse reaction

• Calculate the volume of RNA solution necessary to obtain a total of 3000 ng RNA
• Add SIV to 25 µl
• Add 2.7 µl DNAse buffer and 1 µl DNAseI
• Incubate for 30 minutes at 37 °C
• Add 5 µl inactivation mixture and flip the tubes
• Incubate for 2 minutes in room temperature
• Centrifuge for 2 minutes at 13000 g
• Transfer 2 µl of the supernatant to a new tube and add 18 µl SIV
• Incubate for 10 minutes at 65 °C

cDNA reaction

• Prepare bulk mixture:
  • 5 µl bulk reaction mixture
  • 1 µl RNA primers
  • 1 µl DTT
• Mix 4 µl sample with 3.5 µl bulk mixture
• Incubate for 1 hour at 37 °C
• If qPCR is not to be performed immediately, the cDNA samples should be frozen down at -80 °C

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

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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.

Cake

Batter is prepared following the protocol by Kakemonsen ([http://www.kakemonsen.no/sider/vis.asp?id=1186 1]). Sliced pieces of Malus domestica is inserted into the batter, while sucrose and ground Cinnamomum verum bark is mixed 2:1 by volume and distributed evenly on top of the batter, which is placed in a 26 cm diameter open-top vessel. The mixture is incubated in a dry air oven at ~180 C for 45 min.