Steps

1. Snip 2-4 small leaves, or 1-2 big leaves off each plant.

2. Macerate tissue with blue pestle.

3. Add 800µl extraction buffer

4. Macerate a bit more

5. Vortex 5 sec. (samples can sit at RT here while you process other samples)

6. Spin samples 3 min µfuge, top seed, RT

7. Transfer 600µl supernatant to a new tube containing 600µl isopropanol

8. Invert samples to mix

9. Let them sit at -20ºC for 5-10min

10. Spin 6 min

11. Discard supernatant and wash pellet with 600µl 75% EtOH

12. Dry pellet and add to the pellet 500µl water. Use a blue pestle to “resuspend” (macerate) the pellet.

13. Spin 1 min and transfer the supernatant (avoid the insoluble pellet) to a new tube.

14. Use 1µl per 10µl PCR reaction

Reagent

Extraction buffer

Tris-HCL (pH=7.5) 200 mM

NaCl 250 mM

EDTA 25 mM

SDS 0.5%

This protocol is still under construction!

Steps

1. Put 1.5 mL culture into Eppendorf

2. Spin 14K rpm 30 sec.

3. Discard supernatant, leave 50-100 µL

4. Resuspend bacteria by vortex

5. Add 300 µL TENS, vortex 2-3 sec.

6. Add 150 µL 3M NaOAc pH5.2, mix well gently

7. Centrifuge: 14K rpm, 3 min.

8. Transfer supernatant to new eppendorf

9. Add 1V (0.6 mL) isopropanol, mix well gently

10. Centrifuge: 14K rpm, 3 min.

11. Discard supernatant, wash pellet with 70% EtOH

12. Vacuum dry

13. Dissolve in 30-40 µL TE

Steps

1. Take 50 mL of overnight bacterium culture and transfer to two tubes, centrifuge 8K rpm for 5 min. Discard supernatant, add 2.5ml TE, vortex to resuspend the cells.

2. Add 5 mL Solution B (1%SDS, 0.2N NaOH) and vortex, you should observe the solution become sticky.

3. Add 4 mL Solution C (3M KOAc, pH=4.8) and vortex, you will see the solution turn cloudy.

4. Add 0.5 mL phenol/ chloroform and vortex

5. Centrifuge: 8K rpm for 5 min to remove cell debris

6. Transfer supernatant to a new tube. Add 1V isopropanol and mix well

7. Pellet DNA/RNA in centrifuge for 5 min at 8K rpm

8. Discard supernatant and wash pellet with 70% EtOH, up-side down the tube to remove the last drop of EtOH

9. Resuspend the pellet in 400 µl TE and transfer to an eppendorf tube, add 100 µl 10N Lithium chloride, keep at 4for over 30 min to remove RNA.

10. Centrifuge: 13.2 rpm for 5 min at a microfuge, transfer supernatant to a new eppendorf tube. Add 1V isopropanol and mix well.

11. Centrifuge: 13.2 rpm for 5 min at a microfuge to pellet DNA. Wash pellet with 70% EtOH, and dry the pellet with Spin Vac.

12. Resuspend the pellet in 250 µl TE.

Reagent

Solution B

SDS 1%

NaOH 0.2 N

Solution C

K-acetate 59 g

Acetic acid 22mL

dH2O 150mL

Steps

1. Introduce staining solution by vacuum infiltration (house vac., 5 mins)

2. Incubate samples at 37ºC, 1hr. to overnight

3. Bleach out chlorophyll by adding 70% EtOH (tumble the tubes and change the 70% EtOH several times) (This step takes 3 hours or more)

• High Efficiency Electroporation (Ref: Trends in Genetics 1995, 11:128)

A) Competent Cell Preparation

1. Grow E. coli in 2 ml LB at 37°C overnight.

2. Inoculate 1 ml of cells to 300 ml LB. Shake at 37°C till OD₆₀₀ reaches 0.6 (approx. 2.5 hours). Chill cells on ice for 30 minutes.

3. Harvest cells at 8000 rpm for 5 minutes.

4. Wash twice with 20 ml ice cold water, then wash one more time with GYT buffer.

5. Resuspend cell in 2 ml GYT buffer. Aliquot the cells into Eppendorf tubes and store at –70°C.

B) Electroporation

1. 40-60 µl competent cells are mixed with 1.5 µl ligation mixture, then transfer the mixture to cuvette.

2. Electroporation is performed using MicroPulser (Bio-Rad).

3. After electroporation, add 0.3 ml LB into the mixture and incubate cells at 37°C for 10-20 minutes.

4. The cells were spread on selective agar plates.

GYT Buffer 1 L

10% Glycerol 100 ml

0.125% Yeast Extract 1.25 g

0.25% Trypton 2.5 g

The buffer is aliquoted into small bottle (each 50-100 ml) after complete dissolution.

Revised protocal

A) Competent Cell Preparation

1. Inoculate single colony of E. coli strain (e.g., TG1) to 5 ml LB. Shake culture at 37°C for overnight.

2. Use 2 ml of overnight culture to inoculate a 500 ml LB medium (in 1 L flask). Shake subculture at 37°C for 3 hours (to OD₆₀₀ 0.6-0.9).

3. Transfer cells to sterile centrifuge bottles. Centrifuge at 8000 rpm, 5 minutes, 4°C to pellet the cells.

4. Resuspend cell pellet with a total of 35 ml transformation buffer. Centrifuge at 8000 rpm, 4°C, 5 minutes.

5. Resuspend cells with 10-20 ml of transformation buffer and sit on ice for 1 hour. Then, aliquot competent cells into Eppendorf tubes and store cells in –70°C freezer.

B) Transformation

1. Take a tube of competent cells out of freezer and thaw cells on ice.

2. Use about 100 to 200 µl of competent cells per reaction with less than 10% volume of DNA.

3. Incubate mixture on ice for 30 minutes.

4. Heat shock cells at 42°C for 90 seconds. Add 200 µl LB into the cells and incubate at 37°C for 15-25 minutes.

5. Plate cells on selective plates.

[If required, one may add 20 µl of 10% X-Gal (in N,N’dimethylformamide) and 20 µl of 100 mM IPTG (0.1 g in 4 ml water) to the cells for blue-white selection.]

Transformation buffer 500 ml

MOPS (free acid form, MW = 209.3) 1.05 g

CaCl₂.2H₂O (MW = 147) 6.26 g (85 mM)

D-glucose (Dextrose, MW = 180) 2.5 g

H₂O 425 ml

Glycerol 75 ml (15%)

10 N NaOH 100 µl

pH will be around 6.5. Filter sterilize buffer and store buffer in 4°C.

This protocol is still under construction!

This protocol is still under construction!