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From 2010.igem.org

Contents 1 Preparation of Electrocompetent Cells 2 5.2 Electroporation 3 10/21/10 3.1 Electrophoresis Setup: 4 Electroporation of Agro.Tumefaciens

Preparation of Electrocompetent Cells

See Ausubel et al. (1987) and Miller and Nickoloff (1995) for additional information.

1. Inoculate 500 ml of L-broth with 1/100 volume of a fresh overnight E. coli culture.

2.Grow the cells at 37 °C shaking at 300 rpm to an OD60o of approximately 0.5-0.7 (the best results are obtained with cells that are harvested at early- to mid-log phase; the appropriate cell density therefore depends on the strain and growth conditions).

3.Chill cells on ice for-20 rain. For all subsequent steps, keep the cells as close to 0 °C as possible (in an ice/water bath) and chill all containers in ice before adding cells. To harvest, transfer the cells to a cold centrifuge bottle and spin at 4000 x g for 15 minutes at 4 °C.

4.Carefully pour offand discard the supematant. It is better to sacrifice the yield by pouring offa few cells than to leave any supematant behind.

5.Gently resuspend the pellet in 500 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15 minutes at 4 °C; carefully pour offand discard the supematant.

6.Resuspend the pellet in 250 ml of ice-cold 10% glycerol. Centrifuge at 4000 x g for 15~.minutes at 4 °C; careftiily pour offand discard the supernatant.

7. Resuspend the pellet in -20 ml of ice-cold 10% glycerol. Transfer to a 30 ml sterile Oakridge tube. Centrifuge at 4000 x g for 15 minutes at 4 °C; carefully pour off and discard the supernatant.

8. Resuspend the cell pellet in a final volume~t of ice-cold 10% glycerol. The cell concentration should be about 1-3 x 1010 cells/ml. This suspension may be frozen in aliquots on dry ice and stored at -70 °C. The cells are stable for at least 6 months under these conditions.

5.2 Electroporation

1. Thaw the cells on ice. For each sample to be electroporated, place a 1.5 ml microfuge tube and either a 0.1 or 0.2 cm electroporation cuvette on ice.

2.In a cold, 1.5 ml polypropylene microfuge tube, mix 40 pl of the cell suspension with 1 to 2 ~tl ofDNA (DNA should be in a low ionic strength buffer such as TE). Mix well and incubate on ice for -1 minute. (Note: it is best to mix the plasmids and cells in a microfuge tube since the narrow gap of the cuvettes prevents uniform mixing.)

3. Set the MicroPulser to "Ecl" whefi using the 0.1 cm cuvettes. Set it to "Ec2" or "Ec3" when using the 0.2 cm cuvettes. See Section 4 for operating instructions.

10/21/10

Running Gel on LacZ and T9002 (old/new)

I labled the tubes according to their well number

Tube 1- is the ruler which will have

5 ul of the ruler (nucleid acid sample loading buffer)

5 ul of the (EZ load dye)

15 ul of purified H2O

Note: Before getting the DNA and dye we need to get the gel electrophoresis set up to provide destination of DNA

Electrophoresis Setup:

Got out the BioRad mini sub cell GT electrophoresis Box

We will make the TAE Buffer

Calculations

For 1L TAE (1x) Buffer 20 ml (sox) >> 980 ml 18ohm H2O 500 ml

We put 490 ml of 18ohm H2O into a sterile jar and added 10 ml of TAE Buffer to have a 1x stock concentration

I poured in the TAE Buffer to the box and placed the gel in the Buffer

• Note; Joe removed the gel comb. so ther might be possible contamination made sure the loading wells were placed at the (-) charge side of the box

Tube 2- (B) L10/T30(LacZ 10ul/ T9002 30ul)

added 2 ul of DNA from tube B

added 5 ul of loading dye

added 18 ul of 18ohm H2O

labeled

Tube C- L30/T10

2 ul of DNA

5 ul of loading dye

18 ul of 18ohm H2O

labeled

Tube D- L30/T30

2 ul of DNA

5 ul of loading dye

18 ul of 18ohm H2O

labeled

T9002 (New)

2 ul of DNA

5 ul of loading dye

18 ul of 18ohm H2O

labeled

LacZ(new)

2 ul of DNA

5 ul of loading dye

18 ul of 18ohm H2O

labeled

T9002(old)

2 ul of DNA

5 ul of loading dye

18 ul of 18ohm H2O

labeled

Note: I labeled the old and new T9002 and LacZ plasmid tube w/#’s 5-8 letting me know which tubes I used and the corresponding well

Note: I placed the T9002 and LacZ tubes back in the Fridge and also the unused DNA labled B.D.C in the fridge also.

LacZ(old) The lost tube found 10/21)

2 ul of DNA

5 ul of loading dye

18 ul of 18ohm H2O

labeled

For loading the DNA into the wells, I will be starting with the E2 load ruler/ ladder in well 1 followed by B,CD, 5,6,7

Note: Well 1 will be on the far left sid of the gel this will be the ladder

1 2 3 4 5 6 7 8

-- -- --

-- -- -- -- -- -- -- --

--

--

--

--

Note: JH loaded the wells

wells were loaded at 7:43 pm

voltage is 100 V

time is 30 min. (time was set)

We pulled the gel out when it was finished.

We are not happy with the results, so we put the gel back in and restarted the electrophoresis

100v @ 8:40 pm

Joe added too much ithidium Bromide to gel ant that’s why the gel looks weird

The combs were removed before gel was placed in the buffer with explains why the dye overflowed out of the well.

dgarvey

Electroporation of Agro.Tumefaciens

1. Pipette the DNA samples (up to 5 ~tl) to be elect~opomted into sterile 1.5 ml microfuge tubes; the DNA should be in either water or TE. Place tubes on ice.

2. For each DNA sample to be electroporated, add 1 ml of YM broth to a 17 x 100 tube at room temperature, and place a 0.1 cm electroporation euvette on ice.

3. Thaw the electrocompetent A. tumefaciens cells on ice. For each DNA sample to be electropomted, add 20 gl of eleclxocompetent cells to each DNA sample" gently tap the

4. Set the MieroPulser to "Agr". See Section 4 for operating instructions.

5. Transfer the DNA--cell samples to the eleclropomtion cuvettes and tap the suspension to the bottom of the tube. Place the cuvette in the chamber slide. Push the slide into the chamber until the cuvette is seated betwcen the contacts in the base of the chamber, Pulse once.

6. Remove the cuvette from the chamber and immediately use the YM broth in the " 17 x 100 mm tube to transfer the cells from the cuvette to the tube.

7. Check and record the pulse parameters. The time constant should be about 5 milliseconds The field slxength can be calculated as actual volts (kV) / cuvette gap (cm).

8. Incubate the cells 3 hr at 30 °C, shaking at 250 rpm. Plate aliquots of the electropomted _ i cells on YM agar plates containing the appropriate selective media. Incubate plates for 48 rs at 30 °C.

each in 1.5 ml microfuge tube(sterile)

added 2 ul of “B” directly to thawed Agr.Tum cells (elec.comp)

added 2 ul of “C” directly to thawed Agr.Tum cells (elec.comp)

added 2 ul of “A” directly to thawed Agr.Tum cells (elec.comp)

added 2 ul of “D” directly to thawed Agr.Tum cells (elec.comp)

Note: we agitated and then spun cells down before and after inoculation

Cells were placed back in ice bath.

Electroporator set for Agro

Tub A- 20 ul of cell/DNA-- pulsed once 2.20 kv @ 3.90 ms

GT got 1ml of soc media for me

soc media stored at room temp.

Tub B- 20 ul of cell/DNA-- pulsed once ? kv @ ? ms

Tub C- 20 ul of cells-- pulsed once 2.20 kv @ 1.2 ms

Tub D- 20 ul of cell/DNA-- pulsed once 2.20 kv @ 1.5 ms

20 ul did not seem enough so I added approx 5ul more. pulled w 1ml of soc media

They were stored and labeled in the 4C Fridge.

dgarvey