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==Gel electrophoresis==
==Gel electrophoresis==
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*Add loading dye to the DNA solution. The volume ratio should be 1/5 parts loading dye per part DNA solution (e.g. 2 uL loading dye to 10 ul DNA).
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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.
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Let the gel run for 45 min at 90 V, and if the bands are porly separated, run a little longer. Beware that if the gel runs for a very long time, the smaller bands may run out of the gel.
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
In stead of incubating as described above, the samples are kept at 37 degrees celsius in a water bath for 60 minutes.
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]
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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