Experiments

1. Pool 1 mL culture in 1.5 mL Eppendorf tube by spinning down 1.5 mL at a time and removing supernatant (3-4 times).
2. Resuspend cells in 250 µL of buffer P1 (with RNAse added) in an Eppendorf tube.
3. Add 250 µL Buffer P2 and mix by gently inverting 5 times, leave sit maximum 5 minutes.
4. Add 350 µL Buffer N3 and mix by inverting 5-10 times. Do not vortex.
5. Spin for 10 minutes at 13000 rpm in centrifuge.
6. Apply 800 µL of supernatant to a QIAgen spin column.
7. Centrifuge for 60s, discard flow through.
8. Wash by adding 750 µL Buffer PE (with ethanol added) and spinning for 60s. Remove flowthrough.
9. Spin for an additional 2 min to remove residual PE buffer and discard flowthrough.
10. Transfer to a new Eppendorf tube. Add 50 µL ddH2O to the center of the column, let sit 3 min, then centrifuge 60s. Discard the spin column.

1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm).
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C.

• 10^-1 plate: 100 µL of original
• 10^-2 plate: 10 µL of original + 90 µL of S.O.C.
• 10^0 plate: Spin down remainder @ 13,000 rpm for 1 min, pour off supernatant, resuspend pellet in 100 µL of S.O.C., then plate all 100 µL

1. Larger: 120ml 0.5x TAE buffer + 1.8g agarose, Smaller: 60mL 0.5x TAE buffer + 0.9g agarose. Microwave 40-80s, then 5 µL Midori Green
2. Cast the gel in the gel rig if there is the rubber seal, or tape it if you can't find a rubber sealed one. Put in the comb.
3. Ladder = 15µl “1Kb+ GeneRuler” Ladder + 3µl “Purple Loading Dye” dye.
4. Samples (16x) = 25µl of Digest reaction + 5µl “Purple Loading Dye” dye. You can mix dye into the same tube we did the digest in.
5. Once the gel is cooled, take out the comb and orient the gel so that the wells are on the black side. The DNA will “Run to Red”. Fill the gel rig with more 0.5x TAE until it fills and covers the wells.
6. Add ladder + samples to wells.
7. Run at 100V for 60 minutes.
8. Take gel out and image with “UV365”.

EcoR1 and Cla1:

• 2.5ul rCutSmart Buffer, 0.5µl EcoR, 0.5µl Cla1, 21.5µl DNA.
• Thermocycler: 37C for 40min then 65C for 20 min.

Bsa1

• 5 µL rCutSmart Buffer, 5µL Bsa1, 1 µg DNA (1000/concentration), water to 50 µL
• Thermocycler: 37°C for 5–15 minutes, 80°C for 40 min

Run the Golden Gate mixture in a thermocycler with following program:
30 cycles of:

• 37°C for 1 minute
• 16°C for 1 minute
• 60°C for 5 minutes