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Molecule
Golden gate Protocol
After purification of the PCR product, you can digest your part with EcoRI and PstI using the following Protocol:
| Component | Amount (μl) |
|---|---|
| Purified PCR product | 30 |
| EcoRI | 1 |
| PstI | 1 |
| BsaI | 0.5 |
| NEB buffer 4 (10x) | 4 |
| ddH2O | 3.5 |
| Total Volume | 40 |
| Thermocycler programm |
| 1. 37°C, 12 hours |
| 2. 80°C, 20 minutes |
Important note:
We very much advise you to digest the vector for 12 hours and purify the product on a gel. This significantly reduces the risk of religation of you vector. We usually had no colonies on our negative control plate after ligation with T4 ligase and transformation into DH10B cells.
Since most standard iGEM plasmids contain binding sites of the most common two type IIs restriction enzymes (namely BsaI/Eco31I and BsmBI/Esp3I), we propose using BbsI/BpiI. We have tested this enzyme in various reaction conditions with many different reaction additives (such as ATP or DTT). Although ligase buffer worked best with other type IIs restriction enzymes (in those cases, ligase activity probably was the bottleneck), we had best results with G Buffer (Fermantas) plus several additives using BbsI.
Assembling
For assembling parts that are in Golden Gate standard, we recommend the following protocol:
Ligase Reaction
With the six TAL BioBricks and the fusion enzyme in your reaction tube you now only need the type two restriction enzyme BsmB1 and a T7 Ligase to put all the parts together.
Transform 5 μl of the GATE assembly product into 50 μl of transformation competent bacteria.
Important note
Your cells need to be sensitive to the ccdB kill cassette in our TAL expression vectors! Otherwise also bacteria that have taken up plasmids without the six direpeats will form false positive colonies. We used the DH10B E.coli strain.
In case you want to express your TALE in bacteria, you need to induce the promoter of our prokaryotic expression plasmid with IPTG.
For use in a eukaryotic system, such as HEK 239 cells, perform a midiprep and directly transfect the eukaryotic TAL expression plasmid (or its derivatives pTAL-TF, pTALEN etc.) according to your transfection protocol.
We always used this 8:40 hour thermocycler program to obtain best results. However you can also reduce the number of cycles.
Digestion
Material:
BioLabs ： BstXⅠ R0113S/R0580L
Procedures:
Time-Saver Protocol:
| Components | Volume | |
|---|---|---|
| Restriction Enzyme | 1 μl | |
| DNA | 1 μl | |
| 10× NEBuffer | 5μl | |
| Water | 43μl |
Incubation : 55℃ 5-15min
Material:
Thermo ：T4 DNA ligase
Procedures:
| Linear vector DNA | 20-100 ng |
| Insert DNA | 1:1 to 5:1 molar ratio over vector |
| 10× T4 DNA Ligase Buffer | 2μl |
| 50% PEG 4000 Solution | 2μl |
| T4 DNA Ligase | 5 U |
| Water | to 20μl |
Incubate for 1 hour at 22℃
Material:
Thermo ：Esp3I(BsmBI)
| Components | Volume | |
|---|---|---|
| Restriction Enzyme | 1 μl | |
| DNA | 1 μl | |
| 10× NEBuffer | 5μl | |
| Water | 43μl |
Incubation : 55℃ 5-15min
Material:
Thermo ：T4 DNA ligase
Procedures:
| Esp3I | 0.5-2μl |
| 10×Buffer Tango | 2μl |
| DNA(0.5-1 μg/μL) | 1μl |
| DTT(20mM) | 1μl |
| Water | 16μl |
Incubate at 37℃ for 1-16 hours
Gel extraction
Material:
OMEGA : E.Z.N.A Gel Extraction Kit(Gel Extraction Spin Protocol)
Procedures:
1. Preform agarose gel/ethidium bromide electrophoresis to fractionate DNA fragments
2. When adequate seprartion of bands has occurred, CAREFULLY excise the DNA fragment of interest using a wide, clean, sharp scalpel. Minimize the size of the gel by removing extra agarose.
3. Determine the appropriate volume of the gel slice by weighing it in a clean 1.5ml microcentrifuge tube. Add an equal volume of Binding Buffer(XP2). Incubate the mixture at 55℃-66℃ for 7min or until the gel has completely melted. Mix by shaking or vortexing the tube in increments of 2-3 minutes.
4. Place a DNA column in a provided 2 ml collection tube.
5. Apple 700μl of the DNA/agarose solution to the DNA column, and centrifuge at 10,000×g for 1min at room temperature.
6. Discord liquid and place the DNA column back into the same collection tube.
7. Add 300ml of Binding Buffer(XP2) into the DNA column. Centrifuge at 10,000×g for 1min at room temperature to wash the column. Discard the flow-through and re-use the collection tube.
8. Wash the column by adding 700μl of SPW Wash Buffer diluted with absoluted ethanol. Centrifuge at 10,000×g for 1min at room temperature.
9. Discard liquid and centrifuge the empty DNA column for 2 min at maximal speed(>13,000×g)to dry the column matrix.
10. Place a DNA column into a clean 1.5ml microcentrifuge tube. Add 30μl of distilled water directly onto the column matrix and incubate at room temperature for 1 minute. Centrifuge for 1min at maximal speed to elute DNA.
Important Notes:
1. If the balance liquid turns turbid, incubate it at 37℃ for several minutes.
2. When cutting the gel, keep the UV irradiation time period as short as possible, otherwise DNA 3.would be damaged.
3. For those DNA fragment shorter than 100bp or larger than 10kb, the sol solution volume should be increased as well as the time period for adsorption and elution.
4. The efficiency of eluting DNA from the DNA column is dependent of pH. So make sure that the pH of water is around 8.0.
PCR Clean-Up
Plasmid Extraction
Cell
Construct the gene of our fusion protein: ssDsbA-FP-HL-Lgt-FL-TAL using splicing by overlap extension. We connected ssDsbA-FP-HL-lgt for one part of this fusion protein and FL-TAL for another. Then connect them together.
Protein
Connected ssDsbA-FP-HL-lgt by splicing by overlap extension. Transform, colony picking plasmid extraction and digestion identification; Sequencing results showed accurate construction.
