Particle Bombardment / Gene Gun

Particle bombardment using biolistic gun

(The protocol is described as such it is performed in Plant Transformation Lab)

Preparation of DNA for PDS 1000/He gene gun:

This gun was originally constructed by Sanford et al (1992) and later marketed by BIO-RAD Company.

A.     Micro-carrier preparation

1.      In a 1.5ml microfuge tube, weigh out 3 mg of microparticles (gold particles 1.0 mm) for 6 bombardments.

2.      Add 1ml of 70 % ethanol, fresh prepared.

3.      Vortex on a platform vortexer for 2-3 minutes.

4.      Incubate for 15 minutes.

5.      Pellete the microparticles by spinning for 5 seconds in a microfuge.

6.      Remove the liquid and discard.

7.      Repeat the following steps three times.

a.       Add 1ml of sterile water.

b.      Vortex for 1 minute.

c.       Allow the particle to settle for 1 minute.

d.      Pellete the microparticles by spinning for 2 seconds in a microfuge.

e.       Remove the liquid and discard.

8.      Add 50 ml of sterile water to bring the microparticle concentration to 60 mg/ml.

9.      Store the microparticles at room temperature for up to 2 weeks.

B.    Coating DNA onto microparticles

The following procedure is sufficient for six bombardments; if less bombardment is needed, prepare enough microcarriers for three bombardments by reducing all volumes by one-half. When removing aliquots of microcarriers, it is important to vortex the tube containing the microcarriers continuously in order to maximize uniform sampling.

1.      Vortex the micrcarriers prepared in sterile water (60 mg/ml) for 5 minutes on a platform vortexer to resuspend and disrupt agglomerated particles.

2.      Remove 50 ml (3 mg) of microcarriers to a 1.5 ml microfuge tube.

3.      While vortexing vigorously, add in order:

a.       20ul spermidine (0.1 M)

b.      50 ul CaCl₂ (2.5 M) was added drop by drop on vortex and continued for 2-3 minutes

4.      Allow the microcarrier to settle for 1 minute.

5.      Pellet the microcarriers by spinning for 2 seconds in a microfuge.

6.      Remove the liquid and discard.

7.      Add 140 ml of 70 % ethanol without disturbing the pellet.

8.      Remove the liquid and discard.

9.      Add 140 ml of 100 % ethanol without disturbing the pellet.

10.  Remove the liquid and discard.

11.  Add 48 ml of 100% ethanol.

12.  Gently resuspend the pellet by tapping the side of the tube several times, and then by vortexing at low speed for 2-3 seconds.

13.  Remove 6ul aliquots of microcarriers and transfer them to the center of a macrocarrier. An effort is made to remove equal amounts (500 mg) of microcarriers each time and to spread them evenly over the central 1 cm of the macrocarrier using the pipette tip and Desiccate immediately.

14.  Fix macrocarrier, rupture disk and sieve on the instrument.

15.  Build up 1100 psi pressure to the rupture disk and shoot the DNA on the cells of scueltellum callus.

16.  Culture the tissue on the medium containing manitol (described above) to maintain the osmatic pressure.

Antibiotic Stock Solutions

Antibiotics Stock Solutions (Practically performed working concentration stock solutions for Agrobacterium and E.coli))
Kanamycin (stock 100 mg/ml)

1. Kanamycin-sulphate (1000 mg/10 ml)
2. Double distilled water up to 10 ml volume.
Double distilled de-ionized water was used for stock and final stock was filter sterilized using millipore filters of 0.22µm and stored at –200C in aliquots.

Cefotaxime (Claforan) (stock 100 mg/ml)

1. Cefotaxime (1000 mg/10 ml)
2. Double distilled water up to 10 ml volume.
Double distilled de-ionized water was used for stock and final stock was filter sterilized using millipore filters of 0.22µm and stored at –200C in aliquots.

Ampicilin (stock 100 mg/ml)

1. Ampicilin (1000 mg/10 ml)
2. Double distilled water up to 10 ml volume.
Double distilled de-ionized water was used for stock and final stock was filter sterilized using millipore filters of 0.22µm and stored at –200C in aliquots.

Transformation / Electroporation of Agrobacterium tumefaciens

Transformation of clones in Agrobacterium tumefaciens by electroporation

(The performed protocol using earlier described electro-competent cells of Agrobacterium tumefaciens)

Only 2 ml of each clone was used for electro-transformation by electro cell manipulator 600 (BTX San Diego, California). For electroporation the following protocol was used.

1.      Electroporation cuvettes 1 mm gap were placed on ice. Viols of frozen electro-competent cells of Agrobacterium were allowed to thaw on ice.

2.      1 mg DNA of the recombinant plasmid was mixed with 50 ml of electro-competent cells in the electroporation cuvettes on ice.

3.      The condition for electroporation were set as recommended by the manufacturer:

            Choose mode T                                               2.5 KV

            Set resistance R                                               R5 (129ohm)

            Set charging voltage                                       1.44 KV

4.      The electro-competent cells containing the DNA mixture were transferred to electroporation cuvette.

5.      Pulse was given and 1 ml of liquid LB medium was added immediately, mixed gently and transferred to a 1.5 ml eppendorf tube and incubated at 28°C for 1 hour with vigorous shaking.

6.      200 μl and 400 μl of transformed culture were spread on petri plates containing solid LB medium supplemented with 50 µg of rifampicin and 50 µg of kanamycin/ml, so that only transformed cells should multiply.

7.      When the liquid was absorbed completely the plates were sealed with sealing film and kept at 28 ⁰C for 2-3 days.

8.      At the end of incubation colonies were picked with sterile toothpicks and cultured in 5ml liquid LB medium in 50 ml tube containing 50 µg of rifampicin and 50 µg of kanamycin /ml.

9.      Culture tubes were kept at 28 ⁰C on shaker in Agrobacterium growth room for 48 hours with vigorous shaking.

Transformants were confirmed through PCR.

Preparation of Agrobacterium Electro-Competent Cells

Preparation of electro-competent cells of Agrobacterium tumefaciens

1.      Pick a single colony from a freshly grown plate of Agrobacterium and inoculate into 100 ml LB liquid medium in 250 ml autoclaved flask using sterile toothpick and incubate at 28 ⁰C for 48 hours with vigorous shaking.

2.      Add 5 ml of the 48 hours grown culture re-inoculate into 1 liter flask containing 250 ml of the same LB medium and incubate at 28 ⁰C untill OD₆₀₀ of cells become 0.5-1.0 (10¹⁰ cells/ml).

3.   Transfer the cells aseptically to ice cold 50 ml propylene tube and keep cool on ice for 30 minutes.

4.  Now centrifuge the cells at 4000 rpm for 10 minutes in a bench top refrigerated centrifuge machine at 4 ⁰C. Decant the supernatant and re-suspend the pelleted in 50 ml of sterile cold ddH₂O.

5. Again centrifuge the cells at 4000 rpm in the same centrifuge at 4 ⁰C for 10 minutes and decant the supernatant. Again re-suspend the pelleted cells in 25 ml of sterile cold ddH₂O.

6.      After another wash, re-suspend the cells in 10 ml sterile cold ddH₂O containing filter sterilized cold 10% glycerol. This wash should be repeated.

Finally re-suspend the the cells in 1-1.5 ml filter sterilized cold 10% glycerol, aliquote in 50 ml and store at -70 ^oC.

Transformation of E.coli Cells

Transformation in E. coli DH5- α by electroporation

For electroporation following protocol can be used.

1.      Electroporation cuvetts 2 mm gap place on ice.

2.      Vials of frozen electrocompetent cells of DH5-α allow to thaw on ice.

3.      2 μl of ligation mixtures pipette into Eppendorf tube containing the competent cells and mixe gently with the pipette tip.

4.      The conditions for electroporation are:

Choose mode T                                               2.5 KV

Set resistance R                                               R5 (129 ohm)

Chamber gap                                                   2 mm

Set charging voltage                                       2.45 KV

5.      The electrocompetent cells containing the ligation mixture then transfer to

      electroporation cuvette.

6.      Pulse the mixture and add 1 ml of liquid LB medium immediately, mix gently and transfer to a 1.5 ml Eppendorf tube and incubate at 37 ⁰C for 45 minutes with vigorous shaking.

7.      Spread 100 μl of transformed culture on solid LB medium having antibiotic.

8.      After the liquid absorbed completely, seal the plates with sealing film and keep at 37 ⁰C in incubation for over night.

9.      Pick the Colonies with sterile toothpicks and culture in 3 ml liquid L.B medium containing relevant antibiotic.

10.  Keep culture  tubes at 37 ⁰C in water bath for over night with vigorous shaking.

11.  Isolate plasmid  and checked on 1% agarose gel.

Preparation of E.Coli Competent Cells

Preparation of electro-competent cells of E. coli DH5-α

1.      A single colony from a freshly grown plate of DH5-α (GIBCO-BRL, USA) was picked and transferred into 100 ml LB medium in 1 liter flask and incubated at 37 ⁰C overnight with vigorous shaking.

2.      2.5 ml of the overnight culture was taken and transferred to 250 ml LB in 1000 ml flask and shaken vigorously at 37 ⁰C until O. D⁶⁰⁰ of 0.5-1.0 (10¹⁰ cells/ml).

3.      The cells were transferred aseptically to sterile disposable 50 ml propylene tube.

4.      The culture was cooled by keeping on ice for 10 minutes.

5.      The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in one of volume sterile cold distilled water.

6.      The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in 0.5 volume of sterile cold distilled water.

7.      The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in 0.02 volume of sterile cold distilled water.

8.      The cells were again pelleted by centrifugation at 5000 rpm for 15 minutes and suspended finally in 0.002-0.003 volume sterile 10% cold glycerol.

9.      The cells were stored in aliquots of 50 μl or 100 μl at -70 ⁰C.