DH5α competent cells preparation

1. The day before the preparation, make overnight starters with 100 µL of bacterial cells diluted in 5 mL of LB in a 50 mL Erlenmeyer.
   
2. Prepare buffers 1 and 2 and put them on ice. Put Eppendorf tubes and 50 mL Falcon tubes, and p1000 tips at -80°C for future use.
   
3. The next day, recover your starters and seed 1/60 bacterial cells in 200 mL of LB, then incubate at 37°C and 180 rpm.
   
4. Keep measuring the OD600. When it's between 0,4 and 0,6, recover your cells an pool them in cold 50 mL Falcon tubes.
   
5. Centrifuge tubes at 4000 rpm and 4°C for 10 minutes.
   
6. Discard supernatant and resuspend each Falcon tube in 20 mL of buffer 1. Afterward, centrifuge at 3500 rpm and 4°C for 5 minutes.
   
7. Discard supernatant and resuspend each Falcon tube in 8 mL of buffer 2.
   
8. Take 100 µL aliquots in Eppendorf tubes and stock them at -80°C.
   

Transformation

1. Recover the needed number of competent cells tubes, adding an additional negative control tube.
2. Add DNA (1µL) into the competent cells tubes.
3. Incubate tubes on ice for an hour.
4. Incubate tubes at 42°C for 2 minutes for thermal choc.
5. Leave tubes on ice for 5 minutes.
6. Leave tubes at room temperature for 5 minutes.
7. Add 1 mL of LB per tube, then incubate for 1 hour at 37°C and 180 rpm.
8. Spread cells on Petri dishes and incubate overnight at 37°C.

Monarch PCR and DNA cleanup kit

All centrifugation steps ~13000 rpm in a typical microcentrifuge. This ensures all traces of buffer are eluted at each step.

1. A starting sample volume of 50 μL is recommended. For smaller samples, nuclease-free water can be used to adjust the volume. Add 100 μL DNA Cleanup Binding Buffer to the 50 μL sample.
2. Insert a column into a collection tube, load sample onto the column and close the cap. Spin for 1 minute, then discard flow-through.
3. Re-insert column into the collection tube. Add 200 μL DNA Wash Buffer and spin for 1 minute. Discard flow-through.
4. Repeat Step 5. This step is recommended for removal of enzymes that may interfere with downstream applications.
5. Spin for 1 minute.
6. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute to ensure traces of salt and ethanol are not carried over to the next step.
7. Add 15 μL of hot water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute the DNA.
8. Repeat step 6.

Monarch miniprep kit

All centrifugation steps ~13000 rpm in a typical microcentrifuge. If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve. Store Plasmid Neutralization Buffer (B3) at 4°C after opening.

1. Pellet 1–5 mL bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.

Note: For a standard miniprep to prepare DNA for restriction digestion or PCR, we recommend 1.5 mL of culture, as this is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).

1. Resuspend pellet in 200 μL Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
2. Lyse cells by adding 200 μL Plasmid Lysis Buffer (B2) (blue/green). Invert the tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for 5 minutes.
3. Neutralize the lysate by adding 400 μL of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until the color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
4. Clarify the lysate by spinning for 10 minutes at ~13000pm.

Note:. For culture volumes > 1 ml, we recommend a 5-minute spin to ensure efficient RNA removal by RNase A. Also, longer spin times will result in a more compact pellet that lower the risk of clogging the column.

1. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
2. Re-insert column in the collection tube and add 200 μL of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein, and endotoxin. Centrifuge for 1 minute. Discard flow-through.
3. Add 400 μL of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
4. Centrifuge for 1 minute one more time.
5. Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
6. Add ≥ 15 μL Hot Water (70°C) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Note: Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the DNA Elution Buffer to 50°C prior to use can improve yield.