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Team:Heidelberg LSL/Notebook methods
From 2012hs.igem.org
Methods
Transformation
Transformation is a process in which linear or circular DNA, in other words your designed plasmid, is brought into competent bacteria without the help of bacteriophages. You do this mostly with one of these two intentions: firstly, you want to amplify of your chosen Biobrick-DNA and let transformed bacteria grow it for you is the cheapest and fastest way. Afterwards you have to extract your chosen Biobrick out of your bacteria. Secondly, when you completed your construct in form of a plasmid, you might need to bring it into your final bacteria cells to test if it works.
For amplifying DNA from the registry, we transformed chemocompetent Top10 cells by applying the following protocol:
DNA from Registry Plate: 10 ul water is added to the corresponding well containing the desired BioBrick DNA and DNA is solved by incubation for 15 min at room temperature. 5 ul of DNA solution are added to 50 ul of chemocompetent Top10 cells and incubated on ice for 20 min. Afterwards, a heat shock is performed by incubation the bacteria on 42°C/50 s. After another 2 min on ice, the bacteria are plated onto LB-Amp agar plates.
Inoculation of LB agar plates
Bacteria are cultivated on agar to be amplified and to have the chance to pick single colonies directly from the solid medium.
LB agar is made of LB medium substituted with 100 ug/ml ampicillin and 1.5 % Agar, which makes the LB media solidify.
When plating out transformed bacteria, you should start by carefully labelling the plate with the construct name, date, bacterial strain, antibiotic resistance and your initials and preheat the plate to 37° C. Using a bunsen burner, you then form an inoculation loop out of a small glass pipette. After adding 20-100 ul of bacterial suspension to the plate, you evenly distribute it on the agar plate surface with your glass tool. Now, you can incubate the inoculated LB agar plates at 37 degrees celsius overnight.
Inoculation of liquid LB medium
A bacterial culture in liquid medium is inoculated in order to grow a culture, which is required for a miniprep. Liquid cultures where used during our tests. To inoculate a liquid culture the pellet which has been centrifuged out of another liquid culture is diluted with LB Medium containing the required antibiotics. To inoculate a liquid culture on the base of an agar plate culture you have to pick the colony on the plate with a pipette tip. Then the tip is dropped into the falcon tube with the medium. After the inoculation the falcon or the flask is put in a incubator at 37°C shaking
Qiagen Miniprep
With a Miniprep you extract DNA out of liquid bacterial suspensions such as overnight cultures. There are various companies that offer kits for this process. We followed the instructions of the Qiagen Miniprep kit or the Promega Miniprep kit according to the manufacturer's protocols.
DNA was eluted from the column using either 50 ul of water or TE buffer.
Restriction digest
A restriction digest is the process in which restriction enzymes, small selective DNA-scissors, cut DNA in pieces. These enzymes bind selectively at specific sequences and cut double-stranded DNA either blank or sticky (i.e. they produce up to 4 bp non-paired overhangs). You have to think carefully about the choice of the right restriction enzymes when making up your cloning strategy. For iGEM standard cloning in BBa-format as it was applied here, the following protocol can be applied for all Enzymes used (EcoRI, SpeI, XbaI, PstI).
We used the NEB restriction enzymes and buffers for setting up the digestions. Those were done as follows:
- 1 µg of DNA
- 3 µl of NEB buffer 2
- 3 µl of BSA
- 1 µl of each restriction enzyme used
- Water was added to reach a final volume of 30 ul
After setting up the digestions, the reaction mixtures were mixed briefly, spinned down in a centrifuge (quick run up to 5.000 rpm) and then incubated on a thermomixer heating device for 1 h at 37 °C and stored at -20 °C afterwards. After Digestion, DNA was purified via a qiagen nucleotide removal or gel extraction kit.
SAP digest
Shrimp alkaline phosphatase can be used for dephosphorylation of vector backbone. This ensures, that the vector backbone can neither religate with itself nor with any dephosphorylated insert (that might occur during digestion of the vector backbone) during the ligation process.
For dephosphorylation of vector backbone DNA, 1 unit of FastAP enzyme was added to the digestion mix (after digestion was performed for 1 h) and the mix (including SAP) was incubated at 37 °C for 1 h again.
Oligo design and synthesis
Oligos were designed using WinSerial Cloner and VectorNTI and synthesyzed by Sigma (http://www.sigmaaldrich.com/life-science/custom-oligos.html, 0,025 uM synthesys range).
Gel electrophoresis
Gel electrophoresis is used to display the size of linear DNA fragments and to separate them physically based on this property. In order to do so the DNA is loaded together with a loading dye showing the progress of a running gel onto a agarose gel. The negatively charged nucleotides move towards the positive pole of the applied electric field. The longer a DNA fragment is the larger is the drag caused by the web structure of the agarose gel. To make the DNA bands visible a fluorescent dye like ethidium bromide is added. The EtBr molecules intercale the DNA and glow rose activated by UV radiation. To have a size reference a DNA ladder with fragments of known lengths The gel can be documented with a camera or bands containing required DNA can be cut out easily.
The gel is prepared by adding about 1g/100ml agarose to buffer TAE or TBE, boiling it in a microwave, the addition of ethidium bromide. After that the gel is poured into the gel tray that was sealed before with lab tape and a comb is mounted. The gel tray is transferred to the gel box which is filled with buffer TAE or TBE. Now it can be charged and it is ready to run.
Gel extraction
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Qiagen nucleotide removal kit
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Ligation digest
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Oligo annealing
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Colony PCR
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Sequencing
Sequencing was performed by GATC Biotech (http://www.gatc-biotech.com/en/index.html) using the registry standard sequencing primers VF2 (5’-tgccacctgacgtctaagaa-3’) or VR (5’-attaccgcctttgagtgagc-3’).
X-Gal assay
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ONPG assay
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Use of the photometer
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Use of the plate reader
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Further information
To get further information we highly recommend to visit www.openwetware.org or the pages of the kit distributors.
