Transformation

Miniprep

We built our constructs from digests and ligations of pre-made biobrick parts. Our general conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a summary sheet of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios.

Estimated time

Materials

Biomiga Miniprep Kit

Procedure

1. Harvest the bacterial culture by centrifugation for 1 min at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely.
2. Add 250 µL Buffer A1 (Add RNase A to Buffer A1 before use) and completely resuspend bacterial pellet by vortexing or pipetting
3. Add 250 µL Buffer B1, mix gently by inverting the tube 10 times (do not vortex), and incubate at room temperature for 5 minutes.
4. Add 350 µL Buffer N1, mix completely by inverting/shaking the vial for 5 times and sharp hand shaking for 2 times.
   Note: Incubating the lysate in ice for 1 min will improve the yield.
5. Centrifuge the lysate at 13,000 rpm for 10 minutes at room temperature. If the lysate doesn’t appear clean, reverse the tube angle, centrifuge for 5 more minutes and then transfer the clear lysate to DNA column.
6. Carefully transfer the clear lysate into a DNA column with a collection tube, avoid the precipitations, spin at 13,000 rpm for 1 minute, discard the flow-through and put the column back to the collection tube.
7. Add 500 µL Buffer KB into the spin column, centrifuge at 13,000 rpm for 1 minute. Remove the spin column from the tube and discard the flow-through. Put the column back to the collection tube.
8. Add 650 µL DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 13,000 rpm for 1 minute at room temperature. Remove the spin column from the tube and discard the flowthrough.
9. Reinsert the spin column, with the lid open, into the collection tube and centrifuge for 2 minutes at 13,000 rpm.
10. Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 µL (> 50 µL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again.

Restriction Digest

Ligation

Gel Electrophoresis

Gel Extraction

PCR

PCR Cleanup