From 2009.igem.org

Growing colonies in broth

Prepare 5mL LB + Amp broth in a sterile Falcon tube.
Pick up a colony from the plate by a micropipette tip or sterile toothpick and put it in the falcon.
Incubate at 37C incubator for 14-16 hours.

Miniprep Plasmid Isolation (with Qiagen kit)

1. Centrifuge the falcons at 4000rpm for 4-10 minutes.

2. Resuspend pelleted bacterial cells in 250uL Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf.

3. Add 250uL(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added).

4. Add 350uL Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy.

5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed.

6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through.

7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min.

8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer.

9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32uL (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the center of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min.

Store the minipreps at -20. The concentration obtained can be measured by Nanodrop.

Digestion

For Vector: Mix (to a total of 20 uL):

7-7.5 uL mini-prepped vector DNA 7.5 uL distilled water 0.3 uL restriction enzyme 1 at 20 units/uL 0.3 uL restriction enzyme 2 at 20 units/uL 0.4 uL calf intestinal alkaline phosphatase (CIP) to prevent re-ligation of the vector to itself 2 uL 10x BSA 2 uL 10x appropriate NEB buffer (check from www.neb.com Double Digest Finder)

Insert: Mix (to a total of 10 uL):

7 uL mini-prepped 'vector' DNA 8.2 uL distilled water 0.4 uL restriction enzyme 1 at 20 units/uL 0.4 uL restriction enzyme 2 at 20 units/uL 2 uL 10x BSA 2 uL 10x appropriate NEB buffer

Incubate overnight at 37C.
Gel purify the insert using a Qiagen kit. Elute using 20 uL.
PCR purify the vector (can be gel purified too). Elute in 20 uL.

Ligation (with Roche Rapid Ligation kit)

Mix:

1 uL digested vector 3 uL digested insert

The optimum molar ratio is 1:3, the volumes can be modified according to concentrations of the vector and insert. For sticky end ligations 1:5 ratio can be used.
Complete to 10 uL with 1X reagent 2 of the kit (diluted from 5X with distilled water), Vortex and spin.
Add 10 uL reagent #1.
Add 1 uL reagent #3.
Ligate for 10 minutes at room temperature.
Transform 2 uL of ligation mix in 25 uL DH5alpha competent cells.

Transformation

Thaw competent E. coli on ice. Take 25uL cells into prechilled eppendorfs. Slowly add 2uL plasmid DNA. Do not vortex or make pipetting during the procedure.
Incubate on ice for 30 minutes (or more).
Heat shock at 42C water bath for 30 seconds (timing is critical).
Incubate for 2-5 min on ice.
Add 250 µL SOC medium (~10X volume) and incubate at 37 °C in shaker for 30-60 min.
Spread on LB+Amp plates (or any other selection) using glass beads or spreader.
Incubate overnight at 37°C.

Reference

Harvard Medical School, Silver Lab Protocols: [1] and manuals of the kits mentioned.

Preparation of competent E. coli

* Solutions:

1) LB medium

2) SOB medium

For 500 mL: Tryptone 10 gr Yeast extract 25 gr NaCl 0.25gr

- Dissolve in 450mL and add 5 mL 0.25 KCl (1.86gr KCl in 100mL dH2O)

- Adjust the pH to 7.0 with 5N NaOH.

- Autoclave the medium.

- After autoclaving, add 2.5mL of 2M sterile MgCl2 (18.86 MgCl2 in 100mL)to the media.

3) TB solution

For 100mL Pipes or Hepes (1M) 10 mL CaCl2.2H2O (15mM) 0.221g KCl (250mM) 1.864g

- Dissolve in 80mLof dH2O and adjust pH to 6.7 with KOH.

- Add 1.09g MnCl2.4H2O (55mM) and dissolve completely. Complete the volume to 100mL.

- Filter sterilize the solution, do not autoclave.

* Procedure:

1) Streak DH5alpha on solid LB plates and culture overnight at 37C.

2) Inoculate 10-20 colonies (or 300uL LB culture if liquid medium is used) to 250mL of SOB medium with a loop in a 2L flask.

3)Grow until OD600 reaches 0.6 at 18Cor 37C with vigorous shaking at 200-250rpm.

4) Keep the culture inside the flask on ice for 10 min.

5) Transfer the culture to two seperate sterile centrifuge tubes and centrifuge at 2500g for 10 minutes at 4C.

6) Rsuspend the pellet in 80mL of ice-cold TB and incubate in ice for 10 min, then recentrifuge as previously.

7) Gently resuspend te pellet in 20mL of TB and add DMSO with gentle swirling to a final concentration of 7%.

8) Incubate in ice for 10 min and dispense by 0.4 mL into eppendorf tubes and immediately chill in liquid nitrogen.

9) Store frozen competent cells at -80C.

Reference: Inove et al. 1990.

Machines that we used

1) Centrifuges:

- Hermle Z-233 M-2

- Thermo- Electron Corporation Centrifuge PK110

- Sigma 3K30 Centrifuge

2) Assay Balance: Adventurer OHaus AR2140

3) Electrophoresis and Voltmeter: Orta Doğu Birleşik Teknolojileri LTD Şti

4) Shakers:

- MS2 Minishaker- IKA

- Heidolph MR 3001

5) Water Bath: Memmert-ETG-MBT 250

6) UV: NU72- Flachenstraler

7) Incubator: Ecocell- MMM Group

8) Laminar Flow: Halten

9) Spectrophotometer: Hitachi

10) pH meter: Jenco 6173 pH

11) PCR Machine (Thermal Cyclers)

12) Nanodrop: Thermo Scientific

METU-gene/ Protocols