Molecular Biology


RNA Extraction (added by Elena Loreti)

For 100 mg of tissue (Arabidopsis thaliana, tomato, cereal grains)

1) Homogenize plant material  with 600 µl of extraction buffer*, 7 µl ß-mercaptoethanol and 2.5 µl antifoam

2) Add 84 µl of KCl 3M and keep on ice for 15 minutes

3) Centrifuge  8000 rpm at  4°C

4) Take the supernatant and  add an equal volume of LiCl 8M (to have a final concentration of 4M LiCl); incubate minimum 2 hours at 4°C until overnight

5) Centrifuge 20 minutes at 15000 rpm at 4° C

6) Take the pellet and add 400 µl of  DEPC water,  mix and then add 40 µl of Na-acetate 3M  and mix again

7) Add 450 µl of phenol/chloroform (solution 1:1) and  mix

8) Centrifuge for 5 minutes at 15000 rpm, room temperature

9) Take  water phase (usually the upper one)

10) Add an equal volume of chloroform and  mix

11) Centrifuge for 5 minutes at 15000 rpm, 4°C

12) Transfer the upper phase in a new tube, add -20°C absolute ethanol (optional: add 10 µl Na-acetate 3M)

13) Keep at –20°C for at least 30 minutes

14) Centrifuge for 10 minutes at 15000 rpm, 4°C

15) Take the  pellet and resuspend in DEPC water or TE (usually 50 µl)

 

* Extraction buffer:

Stock solution                          100 mL solution

Tris-HCl 1M                                      5 mL

NaCl 3M                                          10 mL

EDTA 0.5M                                       1 mL

SDS 10%                                         20 mL

DEPC water                                     64 mL


 

Simplified Arabidopsis Transformation Protocol (added by Silvia Gonzali)
(Brief version for those who are familiar with the method)

Steve Clough and Andrew Bent, University of Illinois at Urbana-Champaign.

Our present protocol (Clough and Bent, 1998; modified from Bechtold et al. 1993) is extremely simple. We have found that the MS salts, hormone, etc. make no difference, that OD of bacteria doesn't make much of a difference, that vacuum doesn't even make much of a difference as long as you have a decent amount of surfactant present. Plant health is still a major factor - healthy fecund plants make a big difference! With this method you should be able to achieve transformation rates above 1% (one transformant for every 100 seed harvested from Agrobacterium-treated plants).

1. Grow healthy Arabidopsis plants until they are flowering. Grow under long days in pots in soil covered with bridal veil, window screen or cheesecloth.

2. (optional) Clip first bolts to encourage proliferation of many secondary bolts. Plants will be ready roughly 4-6 days after clipping. Clipping can be repeated to delay plants. Optimal plants have many immature flower clusters and not many fertilized siliques, although a range of plant stages can be successfully transformed.

3. Prepare Agrobacterium tumefaciens strain carrying gene of interest on a binary vector. Grow a large liquid culture @ 28°C in LB with antibiotics to select for the binary plasmid, or grow in other media. You can use mid-log cells or a recently stationary culture.

4. Spin down Agrobacterium, resuspend to OD600 = 0.8 (can be higher or lower) in 5% Sucrose solution (if made fresh, no need to autoclave). You will need 100-200 ml for each two or three small pots to be dipped, or 400-500 ml for each two or three 3.5" (9 cm) pots.

5. Before dipping, add Silwet L-77 to a concentration of 0.05% (500 µl/L) and mix well. If there are problems with L-77 toxicity, use 0.02% or as low as 0.005%.

6. Dip above-ground parts of plant in Agrobacterium solution for 2 to 3 seconds, with gentle agitation. You should then see a film of liquid coating plant. Some investigators dip inflorescence only, while others also dip rosette to hit the shorter axillary inflorescences.

7. Place dipped plants under a dome or cover for 16 to 24 hours to maintain high humidity (plants can be laid on their side if necessary). Do not expose to excessive sunlight (air under dome can get hot).

8. Water and grow plants normally, tying up loose bolts with wax paper, tape, stakes, twist-ties, or other means. Stop watering as seeds become mature.

9. Harvest dry seed. Transformants are usually all independent, but are guaranteed to be independent if they come off of separate plants.

10. Select for transformants using antibiotic or herbicide selectable marker. For example, vapor-phase sterilize and plate 40 mg = 2000 seed (resuspended in 4 ml 0.1% agarose) on 0.5X MS/0.8% tissue culture Agar plates with 50 µg/ml Kanamycin, cold treat for 2 days, and grow under continuous light (50-100 microEinsteins) for 7-10 days.

11. Transplant putative transformants to soil. Grow, test, and use!

For higher rates of transformation, plants may be dipped two or three times at seven day intervals. We suggest one dip two days after clipping, and a second dip one week later. Do not dip less than 6 days apart.

References:

Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C. R. Acad. Sci. Paris, Life Sciences 316:1194-1199.

Clough SJ and Bent AF, 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735-43.

 


 

Competent cells preparation (Rubidium protocol) and Transformation (added by Silvia Gonzali)

(From ROB HALLEWELL OF CHIRON CORP.)

1. Grow single colony at 37°C in 5 ml LB-broth O.N.
   
2. Transfer to 100 ml prewarmed LB-broth and grow until OD550 = 0.5 (approximately 2-2.5 hours). Vigorous aeration is necessary: rotation should be 250-350 rpm, and culture volume should not exceed 40% of flask volume.
   
3. Chill cells on ice, transfer to sterile Falcon tubes (2 x 50 ml) and spin for 10 minutes at 2500 rpm in pre-chilled refrigerated centrifuge.
   
From now on, the cells must remain at 0°C: therefore keep containers on ice, use chilled pipettes, and if possible work in the cold room.
   
4. Take up the pellets (with brief vortexing) in ice-cold “Transformation buffer I” using 30 ml Tf.B.I per 100 ml of starting culture.
   
5. Incubate 120 minutes on ice.
   
6. Spin for 5 minutes at 2500 rpm, resuspend as gently as possible in ice-cold “Transformation buffer II” adding 4 ml Tf.B.II per 100 ml of starting culture.
   
7. Aliquot 100 microlitre quantities into sterile ice-cold Eppendorf tubes, put into chilled holder and freeze at or below -70°C.
   
The competent cells can now be stored at least 3 months without marked reduction in their transformation potential.
   
8. For transformation: thaw aliquot at room temperature until just melting and then put on ice for 5-10 minutes (can leave up to 1 hour).
   
9. Add DNA in a volume of TE buffer less than 25 microliters. Don’t use more than 0.1 microgramme DNA per 100 microlitre of cells.
   
10. Mix and leave on ice 20 minutes.
   
11. Heat-shock 90 sec. at 42°C or 120 sec. at 37°C.
   
12. Return to ice for 1-2 minutes, then add 5 volumes LB-broth + glucose (at room temperature).
   
13. Incubate at 37°C for 2 hours.

14. Centrifuge at 10.000 x g for 1 min and resuspend pellet in 150 microlitre LB-broth + glucose.
   
15. Spread 50-100 microlitre aliquots on selective nutrient agar plates.

16. Incubate at 37°C O.N..

Transformation buffer I: 30 mM Potassium Acetate, 50 mM MnCl2, 100 mM RbCl, 10 mM CaCl2, 15% (w/vol) glycerol. Mix and pH down to 5.8 with 0.2 M Acetic Acid (throw out and start over if you overshoot). Filter-sterilize and store at 4°C.

Transformation buffer II: 10 mM NaMOPS (pH 7.0), 75 mM CaCl2, 10 mM RbCl, 15% glycerol. Filter-sterilize and store at 4°C.

LB-broth: for 500 mL: 5 g Tryptone + 2,5 g Yeast extract + 5 g NaCl

LB-broth + glucose: 10 mL LB-broth + 200 μl 20% glucose

 


 

Rapid DNA preparation (added by Silvia Gonzali)

(from: EMBO COURSE “Practical Course on Genetic and Molecular Analysis of Arabidopsis”)

A crude and rapid extraction from a small leaf disk is sufficient to isolate genomic DNA suitable for PCR amplification. The present protocol is based on the method described by Edwards et al. [Nucl. Acids Res. 19, 1349 (1991)]. All steps are performed at room temperature.

1. Use the lid of a 1.5 ml Eppendorf tube to pinch out a disc from a young leaf into the tubea.
2. Use a small pestle to grind the leaf material in the tube without buffer for approx. 15 seconds.
3. Add 400 µl of extraction bufferb and vortex for 5 secondsc.
4. Spin 1 minute at full speed in a microfuge to pellet the debris.
5. Transfer 300 µl of the supernatant into a fresh 1.5 ml Eppendorf tube  (avoid taking debris from the pellet).
6. Add 300 µl isopropanol, mix and leave at room temperature for approx. 2 minutes.
7. Spin 5 minutes at full speed in a microfuge to pellet the DNA.
8. Remove all the supernatant and dry the pellet gently (for istance by incubating the open tubes at 37°C for a few minutes). Do not let the pellet get too dry, otherwise it will be very difficult to redissolve the genomic DNA.
9. Add 100 µl TE and dissolve the pellet by gentle shaking (do not vortex).

Notes

a It is important to avoid using too much starting plant material. In contrast, a smaller piece of leaf or a single cotyledon can be used, in which case all volumes should be reduced by 50% in the subsequent steps of the extraction procedure.
b Extraction buffer: 200 mM Tris-HCl pH 7.5 (pH 8.0 is ok), 250 mM NaCl, 25 mM EDTA, 0.5% SDS.
c At this stage, the samples can be kept at room temperature for up to one hour until all the samples have been extracted.

N.B. Set up a 20 μl PCR reaction mix containing 1 μl of genomic DNA from the miniprep method described above