virus induced gene silencing

WORKSHOP AND HANDS ON TRAINING ON

VIRUS INDUCED GENE SILENCING December 1-14, 2010

Sponsored by Department of Science and Technology Government of India New Delhi

DEPARTMENT OF GENETICS AND PLANT BREEDING C. CHARAN SINGH UNIVERSITY MEERUT-250 004

Experiment 1 Selection of candidate gene, search for homology and designing of oligos A. Selection of candidate gene Select a candidate gene with following features: 1. Candidate gene should already be cloned in a model plant system or in a related crop species. 2. Clear/known phenotypic characteristics. 3. Known developmental stages on which candidate gene expressed. 4. Known nucleotide sequence i.e. EST, mRNA or cDNA sequences. B. Search for homology 1. Download EST sequence of gene of your interest. Use EST sequence of your candidate gene for BLAST search to identify orthologues/paralogues in the crop of your interest (Fig. 1).

Fig 1. Flow diagram of oligo designing for VIGS.

Experiment 2 Preparation of Chemically Competent Cells (DH5α) A. Materials and Equipment Beckman Table Top Centrifuge 30 ml Glass Corex Tubes Falcon 2059 Polypropylene Tubes 250 ml Plastic Bottles 37oC Shaker 42oC Water Bath 37oC Incubator Dry Ice/LN2 B. Solutions LB Broth 10 grams Tryptone 5 grams Yeast Extract 0 grams Sodium chloride Water to 1 liter. Autoclave LB Agar 10 grams Bacto Tryptone 5 grams Bacto Yeast Extract 10 grams Sodium Chloride 15 grams Bacto Agar Water to 1 liter Autoclave Cool to 55oC. Add antibiotic Pour into plates 2M CaCl2 22.198 grams CaCl2, Anhydrous Water to 100 ml Filter Sterilize Glycerol Ethanol Ampicillin

HAZARDS Ethanol Flammable liquid and vapor. Poison. Wash thoroughly after handling. Remove contaminated clothing and wash before reuse. Use only in a well-ventilated area. Ground and bond containers when transferring material. Use spark-proof tools and explosion proof equipment. Avoid contact with eyes, skin, and clothing. Empty containers retain product residue, (liquid and/or vapor), and can be dangerous. Keep container tightly closed. Avoid contact with heat, sparks and flame. Do not ingest or inhale. Do not pressurize, cut, weld, braze, solder, drill, grind, or expose empty containers to heat, sparks or open flames. Dry ice PROTECTION Lab coat Eye Protection Gloves Absorbent bench paper Hood WASTE Ethanol must be disposed of into appropriate hazardous chemical waste containers. Bacterial broth must be bleached or autoclaved prior to discard. C. Protocol 1. Streak out DH5α from glycerol stock on LB agar plate. 2. Pick an isolated colony and grow a 5 ml culture in LB (without antibiotic) overnight at 37°C with shaking @ 200 rpm. 3. Next day, dilute overnight culture 1:100 in LB broth (without antibiotic; in 500ml). 4. Grow culture to an OD600 of 0.3-0.5 at 37°C with shaking. 5. Takes approximately 2.5 hours, but check often after reaching 0.2. 6. Spin down cells in sterile 250ml bottles at 4000 rpm, 4°C for 10 minutes. 7. Resuspend in 500ml ice-cold 50mM CaCl2. 8. Incubate on ice for 20 minutes. 9. Pellet cells by spinning at 4000 rpm for 10 minutes at 4°C. Resuspend cells in 12 ml of the following STERILE solution: 0.53 ml 2M CaCl2 (stock concentration) 1.675 ml 100% glycerol 10.09 ml sterile ddH2O 11. Dispense in 50 µl aliquots of competent cells into 1.5 ml eppendorf tubes and quickfreeze dry ice or liquid nitrogen. 12. Store at -80°C 13. Use 50 µl per transformation.

Experiment 3 Transformation of chemically competent cells with α, β, γ-PDS, γ-MCS plasmids 1. Use a stock of plasmid DNA solution (100 ng/µl) for transformation. 2. Thaw competent cells on ice. 3. Add 5 ml (50 pg) control DNA to one polypropylene tube (Falcon 2059) containing 100 ml competent cells. Move the piptette through the cells while dispensing. Gently tap the tube to mix. 4. Incubate cells on ice for 30 minutes. 5. Heat-shock cells 90 seconds in a 42oC water bath; do not shake. 6. Add 1 ml of SOC (no antibiotic) that was kept on room temperature. 7. Shake at 200 rpm and 37 oC for one hour. 8. Plate out 100 µl and 10 µl of the above solution on an LB agar plate containing 100 mg/ml ampicillin. 10. Incubate the plates overnight at 37oC upside-down.

Experiment 4 Preparation of Electro Competent Cells A. Materials and Equipment Beckman Table Top Centrifuge 30 ml Glass Corex Tubes Falcon 2059 Polypropylene Tubes 250 ml Plastic Bottles 37oC Shaker 42oC Water Bath 37oC Incubator Dry Ice/LN2 B. Solutions LB Broth 950 ml of autoclaved ddH2O 10 g bacto-tryptone 5 g bacto-yeast extract 10 g NaCl. 15 g bacto-agar, to solidify. Mix, autoclave. LBamp: add 1 ml of the 100 mg/ml stock ampicillin to each liter of cooled broth or cooled (not solidified) agar. Swirl to mix. SOC medium 950 ml of autoclaved ddH2O 20 g bacto-tryptone 5 g bacto-yeast extract 0.5 g NaCl Mix until dissolved. Also, make up a 1M solution of glucose (18 grams of glucose in 90 mls of ultrapure water). Autoclave both solutions separately. Allow the medium to cool to 60 degrees or less, then add 20 ml of the sterile glucose solution. C. Protocol 1. Streak E. coli DH5α from the frozen stock onto a fresh LB plate. Incubate plate upsidedown at 37oC overnight. 2. Use a single colony to inoculate 10 ml of LB broth. Place on shaker at 200rpm, at 37oC overnight (primary culture). 3. Inoculate 1 liter of fresh LB broth with 10 ml of the fresh overnight LB culture (secondary culture). 4. Grow cells at 37oC with vigorous shaking to an A600 of 0.5-1.0 (for DH5 , this is about 5 hours).

5. To harvest, chill the flask on ice for 15-30 minutes, then centrifuge in a cold rotor at 4000 rpm for 10 minutes. 6. Remove as much of the supernatant as possible. Resuspend pellets in a total of half volume of ice chilled autoclaved ddH2O. Centrifuge as in step 5. 7. Repeat step 6. 8. Resuspend pellet in 20 ml of ice chilled autoclaved 13% glycerol. Centrifuge as in step 5. 9. Resuspend to a final volume of 2 or 3 ml in ice chilled autoclaved 13% glycerol. 10. Divide the cell suspension into aliquots of 50 µl per tube in eppendorf tubes. Dip the tubes into liquid nitrogen to freeze, store at -80 until used. Cells will be good for 6 months to a year.

Experiment 5 Transformation of electro competent cells with α, β, γ-PDS, γ-MCS plasmids 1. Remove 1 tube of frozen competent cells from the freezer for each transformation that will be done. Thaw the cells slowly on ice. Mix gently to resuspend, and return to the ice. 2. Pre-chill required number of electroporation vials on ice. Label tops. 3. Mix DNA and 50 microliters of chilled cells in cold eppendorf tubes. Transfer to chilled electroporation vials, knock sharply on the desk to remove any bubbles. Return vials to ice. 5. Place the electroporation vial containing the DNA and bacterial cells into the chamber (dry off the bottom with a kimwipe, first), slide it into place so that contact is made with the electrodes, then push both buttons on the apparatus at the same time, until the buzzer sounds. 6. AS QUICKLY AS POSSIBLE: slide the electroporation cell out of the chamber, take off the lid, and add 1 ml of chilled SOC medium. Mix briefly, then return the cells to the ice until you have finished electroporating all your samples. 7. Transfer samples to microfuge tubes, incubate the tubes for 60 minutes on the shaker at 37oC. 8. Plate out 100 µl and 10 µl of the above solution on an LB agar plate containing 100 mg/ml ampicillin. 9. Incubate the plates overnight at 37oC upside-down.

Experiment 6 Growing of bacterial culture (viral plasmid DNA transformed DH5α) A. Preparation of media LB broth: 200 ml x 4 Suspend 5 gm of LB broth in 200 ml of dH2O. Sterilize by autoclaving at 15 lbs for 15 min. B. Inoculation of transformed DH5α Four colonies of each viral plasmid transformed bacteria (α plasmid, β plasmid, γ-PDS plasmid, γ-MCS plasmid) from over night grown LB agar plates were picked by autoclaved pipette tips and were used to inoculate 200 ml of LB broth in laminar air flow chamber. All four conical flasks containing above transformed bacteria were placed in incubator shaker at 200 rpm and at 37oC for overnight.

Experiment 7 Isolation of Plasmid DNA (Midi preparation) A. Material required Qiagen kit: Buffer P1, Buffer P2, Buffer P3, Buffer QBT, Buffer QC, Buffer QF, RNase A, Qiagen columns. Isopropanol Ethanol (70%) Centrifuge set at >20,000 rcf (g) and 4oC. B. Protocol (using Qiagen midi kit) 1. All RNase A solution and LyseBlue reagent were added to buffer P1. 2. Buffer P3 was pre-chilled at 4 oC. 3. Bacterial culture, harvest and lysis:  Pellet the over night grown culture at 6000 x g for 15 min at 4oC.  Homogenously resuspend the bacterial pellet in 4 ml of buffer P1.  Add 4 ml of buffer P2, mix thoroughly by inverting 4-6 times, and incubate at RT for 5 min.  Add 4 ml of buffer P3, mix thoroughly by inverting 4-6 times, and incubate on ice for 15 min. 4. Bacterial lysate clearing:  Centrifuge the tubes containing lysate at >20000 x g for 30 min at 4oC.  Re-centrifuge the supernatant at >20000 x g for 15 min at 4oC. 5. Bind, wash, and elute plasmid DNA on Qiagen column:  Equilibrate Qiagen column 100 by applying 4 ml buffer QBT and allow column to empty by gravity flow.  Apply the supernatant (step 4) to the Qiagen column and allow it to enter the resin by gravity flow.  Wash the Qiagen column with 10 ml buffer QC twice. Allow buffer QC to move through the Qiagen column by gravity flow.  Elute DNA with 5ml buffer QF into clean 15 ml round bottom centrifuge tube. Note: Check the quality of plasmid DNA obtained by running on 0.8% agarose gel (loaded 10µl from 5ml) at 80V for 2hr.

6. Precipitate, wash and redissolve plasmid DNA  Precipitate DNA by adding 3.5ml RT-isoproponol to the eluted DNA and mix. Keep it for 1hr at 4oC. Centrifuge at >15000 x g for 30 min at 4oC. Carefully decant the supernatant.  Wash DNA pellet with 2ml RT-70% ethanol and centrifuge at >15000 x g for 10 min at 4oC. Carefully decant the supernatant.  Air-dry the pellet for 5-10 min and redissolve DNA in 500µl of TE buffer. 7. Checking of concentration of plasmid DNA  1µl of plasmid DNA was picked from 500µl stock DNA and diluted with 199µl of loading buffer (3 T.E. : 1 6X loading dye).  5µl and 10µl of the above 200µl diluted solution was loaded in 0.8% agarose gel. Gel was run at 80V for 2 hr.

Experiment 8 Preparation of γ-PDS plasmid DNA A. Digestion of the γ-PDS plasmid DNA The plasmid DNA was digested with Pac1 and Not 1 separately 

For digestion with Pac1 following reaction was set for 250µl (as per availability of the restriction enzyme) final volume: H2O – 97.5 µl DNA – 100 µl (1µg/µl) 10X buffer – 25µl BSA – 2.5µl Pac1 restriction enzyme – 25µl (stock-10U/µl) Incubated in PCR thermal cycler overnight at 37 oC.

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After digestion with Pac1 the DNA was precipitated with the following protocol: DNA – 250µl (100µg) 3M Sodium acetate (pH 5.2) – 25µl (1/10 of the total volume) 100% chilled ethanol – 550µl Keep at 4oC for 10 min. DNA precipitates. Centrifuged at 14000 rpm for 10mins. Pellet was placed in 70% ethanol for 30 mins or more at 4oC. Centrifuged at 14000 for 5mins. Decant off the supernatant and dry (5-10 mins). Dissolve the pellet in water (volume required for second digestion reaction i.e 240µl).

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For digestion with Not1 following reaction was set for 300µl (as per availability of the restriction enzyme) final volume: DNA dissolved in H2O – 240µl 10X buffer O with BSA – 30µl Not1 restriction enzyme – 30µl (stock-10U/µl) Total Volume – 300µl Incubated in PCR thermal cycler overnight at 37 oC.

B. Checking of digestion of γ plasmid DNA

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After completion of digestion, add 60µl of 6X loading dye to digested plasmid DNA reaction and incubate the samples at 60oC for 5mins (to reduce the chances of re-ligation of PDS insert into vector). Note: Here, sample volume is 360µl. To loading of large volume of DNA, joint 8-10 tooth of comb by cello tape and cast the gel (0.8% agarose gel). Transfer the heated samples on ice and load the samples in 0.8% agarose gel. Run the gel overnight at 50 volts (25mAmp) or at 70 volts if power supply showed