Laboratory procedures

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

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.