From 2007.igem.org

Maxiprep transformation

Make sure that the incubator (30/37C) and water bath (42C) are ON

Make sure required antibiotic plates are present. Check the antibiotic resistance on the plasmid map in Vector NTI.

Take the DNA out of -20 frig, let it thaw

Thaw the competent cells on ice for 7-8 min.

Add 1.0 µl of DNA (about 10ng) into the liquid (Don’t vortex). Tap the sides of the tube to mix.

Incubate the cells on ice for 30 min

Heat shock the cells for EXACTLY 30 sec at 42 C water bath.

Place on ice for 2 min.

Add 0.9ml of 37C S.O.C medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)

Shake the tubes at 37 C, 280 rpm for 60 min or 30 C for 90 min

Incubate upright for 20 min., then upside down overnight (12-14 h) at 37 C or 16-18h at 30C.

Can leave the cells in the incubator for up to 18 hours but no more

Maxiprep growth culture

Calculate the approx. S/N ratio of the transformation.

Decide on number of tries to be setup accordingly. Pick 12 tries if the S/N ratio is good (5:1), more otherwise.

Aliquot X ml (X = Y number of tries * 3.5, where Y = number of tries.) of TB into a clean (autoclaved) beaker or flask. Lb can be used if TB is not available.

Add X µl of appropriate antibiotic, i.e. a 1:1000 dilution to the above solution. The correct antibiotic should be added (same as the plate from which the colonies are picked.)

Take Y clean and autoclaved 14 ml polystyrene tubes (ones with thw white cap) and place them on a rack. Label them clearly. Aliquot 3.5 ml of TB/LB into each 14 ml tube.

Use a sterile wooden applicator (autoclaved) or pipette tip to carefully pick an individual colony and dip the colony end of the applicator / tip into a 14 ml tube. Repeat this Y times using a fresh applicator each time. Make sure to pick a single colony per try.

Place the tubes in the 37C shaker at 280-300 rpm and grow the cells for 12-14 hours. The tubes should be murky after the overnight growth.

Maxiprep DNA extract

Transfer 200ml of the overnight culture of plasmid cells into a clean, autoclaved 200ml centrifuge bottle (1 per plasmid). Pellet for 5 min. Decant all the liquid and add the remaining 200ml of each plasmid culture into the corresponding bottle. Make sure not to mix up the plasmids. Spin down again for 5 mins. And throw away the supernatant.

Add 15ml of Resuspension solution to the cell pellet and vortex the bottle. No cell clumps should be observed.

Add 23ml of Cell Lysis solution. Do not vortex. Mix gently by swirling the bottle. The solution should become slightly clear and very viscous at this stage.

Add 15ml of Neutralization solution. Do not vortex. Mix gently by swirling the bottle. A white precipitate will appear.

Spin down the cells for 25 mins. While this is being done, mix the bottle labeled ‘matrix solution’ well by vortexing till you have a homogenous solution.

The supernatant at this stage contains the plasmid DNA. So, do not discard the supernatant. Carefully transfer the supernatant into a fresh 200 ml bottle. Do not transfer any of the precipitate. Make sure not to mix up the supernatants and label all bottles at each stage.

Add 10ml of the matrix per bottle. Swirl and mix for 30 sec. Centrifuge for 5 min. The matrix binds the DNA and settles at the bottom.

Throw out the supernatant. Resuspend the matrix in 25 ml of wash buffer. Centrifuge for 5 min. Make sure that the wash buffer bottle is marked ‘ethanol added’. If not, add 100% ethanol to fill the wash buffer bottle. Assemble the filters. Place each filter provided in the kit in a 50ml centrifuge tube (blue cap, also provided in the kit). Label the tubes.

Throw out the supernatant. Resuspend the wash buffer in 15 ml of the wash buffer and mix thoroughly. Transfer the solution to the corresponding filter/tube from the above step.

Centrifuge for 5 min.

Remove the filter and throw out the solution. Place the filter back in the same tube. Add 10 ml of wash buffer and centrifuge for 5 min.

Remove the filter and place it in a new 50 ml centrifuge tube (provided in the kit). Label the tube and add 4 ml of EB (elution buffer) to the filter. Let it stand for 3 min at RT.

Centrifuge for 5 min.

Discard the filters from each tube. Add 222 µl of 5M NaCl to each tube. Mix well by swirling gently. Add 8ml of ice cold 100% ethanol to each tube. Centrifuge at speeds > 10000g for 15 min.

You will see a translucent white DNA precipitate at the bottom. Carefully throw away the supernatant without disturbing the pellet.

Add 10 ml of 70% ethanol to each tube. Centrifuge at speeds > 10000g for 15 min.

Once again, you will see a translucent white DNA precipitate at the bottom. Carefully throw away the supernatant without disturbing the pellet.

Air dry the pellet (leave it on your bench for 10-15 min.) Don’t leave the pellet for a longer duration as it might be difficult to resuspend.

Resuspend the pellet in 1.5 ml of EB.

Measure the concentration and aliquot into labeled 2ml Eppendorf tubes.

Store the DNA at -20C.

Ultracentrifuge, 120mm

6000, 6682

10000, 8626

15000, 10564

20000, 12199

Ultracentrifuge, 65mm

20000, 16575

15000, 14354

Centrifuge, 185mm

6000, 5381

10000, 6947

Equipment to bring to measure OD:

p200, p20, tips

Blanking solution, alcohol wash bottle, DI water wash bottle.

Tray of samples/unknowns and required number of Eppendorfs.