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
Mastermix for real time PCR
Coming soon...
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.
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