Protocols
No. 5 - Ligation
How to assemble DNA biobricks
Materials
Ligation mixture:
For 1 ligation reaction
• 2 µL 10x T4 ligase buffer
• 1 µL T4 ligase (add last!)
• 5 µL PCR product (cut) of each brick which is to be ligated – or 1 part plasmid and 5 part bricks
Protocol
1. Prepare the ligation mixture and mix by pipetting up and down
2. Leave the mixture over-night at 17°C
3. Test ligation using TAQ-PCR and run test gel afterwards in order to check that the PCR product has the right size
How to extract and purify DNA from gel
Important remarks
All steps should be carried out at room temperature.
All centrifugations should be carried out in a table-top microcentrifuge at >12000x g
Materials
• Binding buffer
• Wash buffer (diluted with ethanol)
• Elution buffer
Protocol
1. Weigh a 1.5 µL tube
2. Excise gel slice containing the DNA fragment using a clean scalpel (cut as close to the DNA as possible)
3. Place the gel slice into the pre-weighed tube and record the weight of the gel slice
4. Add 1:1 volume of binding buffer to the gel slice (e.g. add 100 µL of binding buffer for every 100 mg of agarose gel)
5. Incubate the gel mixture at 50-60°C for 10 min. or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes. The color of the solution should be yellow. If the color of the solution is orange or violet add 10 µL 3M sodium acetate , pH 5.2 and mix. The color will then turn yellow.
6. Transfer up to 800 µL of the solubilized gel solution to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place column back into the same collection tube
7. Add 700 µL of Wash buffer to the column. Centrifuge for 1 min. Discard flow-through and place the column back into the collection tube
8. Centrifuge the empty column for an additional 1 min. to completely remove residual Wash buffer This step is essential to avoid residual ethanol in the purified DNA solution
9. Transfer the column into a clean 1.5 mL microcentrifuge tube. Add 50 µL of Elution buffer to the center of the column membrane. Centrifuge for 1 min.
10. Discard the column and store the purified DNA at -20°C
"
