Workflow

1. Miniprep/Nanodrop
2. Digest
3. Gel
4. Ligation
5. Transformation, Plate
6. Colony PCR (Screening)
7. Gel
8. Inoculate correct colony to a liquid culture.

Materials:

Miniprep: grown culture, microcentrifuge, 2 1.5mL microcentrifuge tubes, mini column and collection tube, Solution I, Solution II, Solution III, HBC Wash Buffer, DNA Wash Buffer, Elution Buffer, micropipette and tips

Nanodrop: nanodrop machine, miniprepped DNA, Kimtech wipes, micropipette and tips

Digest: miniprepped DNA, dH₂O, 10X RE-Mix, standard restriction enzyme, micropipettes and tips

Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA

Ligation: vector, parts 1 and 2, ligase buffer, ligase, Antarctic phosphatase, microcentrifuge tube, ice, micropipette and tips

Transformation: ice, ligation mixture, competent cells, incubator, LB media, microcentrifuge tubes, micropipette and tips Plate: agar plate, micropipette and tips, beads

Colony PCR: dH₂O, buffer, VF₂, VR, Q5 polymerase, dNTP, DNA dilution, micropipette and tips, PCR tubes, thermocycler, ice
Gel: agarose gel (make one if necessary), 1X TAE Buffer, power supply, chamber and electrodes, ladder, micropipette and tips, DNA

Inoculate: LB media, dilution, micropipette and tips

Protocol:

   1. Miniprep (using Omega protocol)      1.1 Grow 1-5mL culture overnight in a 10mL-20mL culture tube.      1.2 Centrifuge at 2500xg for 5 minutes at room temperature. Decant or aspirate and discard the culture media.           1.2.1 Original protocol called for 10,000xg for 1 minute, but the speed and time above seemed to produce better results.      1.3 Add 250uL of Solution I mixed with RNase A (pre-added). Vortex to mix thoroughly. Transfer the suspension into a new 1.5mL microcentrifuge tube.      1.4 Add 250uL of Solution II. Invert several times until you get a clear lysate.           1.4.1 Once Solution II is added, do not let it sit for more than 5 minutes!      1.5 Add 350uL of Solution III. Invert several times until a white precipitate forms. Centrifuge at 13,000xg or 17,900rcf for 10 minutes. A compact white pellet should form at the bottom of the tube.      1.6 Insert a mini column into a 2mL collection tube.      1.7 Transfer the clear supernatant into the mini column using a micropipette. Centrifuge at the maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.           1.7.1 Be careful not to get any parts of the pellet! Tilt at an angle with the pellet at the top when micropipetting is advisable.           1.7.2 Think about what you are discarding versus what you want to keep!      1.8 Add 500uL of the HBC Wash Buffer diluted in isopropanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.           1.8.1 All wash buffers will be centrifuged for 1 minute.      1.9 Add 700uL of the DNA Wash Buffer diluted in ethanol. Centrifuge at maximum speed (13,000xg) for 60 seconds. Discard the filtrate and reuse the collection tube.      1.10 Centrifuge the empty mini column at the maximum speed (13,000xg) for 2 minutes to remove the ethanol.      1.11 Transfer the mini column to a nuclease-free 1.5mL microcentrifuge tube.      1.12 Add 50uL of Elution Buffer (or sterile deionized water). Let it sit in room temperature for 60 seconds. Centrifuge at maximum speed (13,000xg) for 60 seconds.      1.13 Store eluted DNA at -20℃.