Protocols

1. Kenaf protoplast isolation and transfection

Day 1

• Prepare enzyme solution by dissolving 0.32g of cellulase (0.8%) and 0.12 g of Macerase (0.25%) in 40 ml of enzyme solution. Filter sterilize using 0.22μ filter.
  
• Sterilize 2 grams (can go up to 3 grams) of kenaf leaves in 0.8% Clorox for 10 minutes.
  Rinse with autoclaved water thrice in laminar hood.
  
• Cut the leaves into 1 mm strips and incubate in enzyme solution on shaker 10 g (speed 1-2 in Heidolph shaker) for 16 hrs.

Day 2

• Release the protoplasts on the dish by gently pipetting.
  
• Pass the protoplasts through a sterile sieve and then pass the protoplasts thro 70μ nylon screen (Falcon) and remove the debris. Transfer the filtrate to falcon tube.
  
• Spin at 750 rpm for 7 minutes,store the pellet.Further spin the supernatant at 850 rpm 7 min and pool the pellets and add wash solution (40 ml).
  Spin at 750 rpm for 5 min. Repeat wash once more with 40 ml of wash solution.
  
• Re-suspend the protoplasts in X ml of wash solution( For each sample, 1-2ml of wash solution.). Transfer the protoplast mix into a new 15ml falcon tube. Spin at 750 rpm for 5 min, then remove the supernatant. Gently resuspend the pellet.
• Protoplast transfection:
  
  Add 10 μg of viral RNA in resuspended protoplasts, mix gently.
  Add 100 μl of 40 % PEG 3000 in 3mM CaCl2 into the protoplasts and mix. (Protoplasts might form clumps. To avoid clumping, add PEG slowly little by little at the side of the wall of falcon tube.)
  Add 4.5 ml of wash solution,resuspend the protoplast well and incubate on ice for 30 min – 1 hr.
  Spin down at 750 rpm 5 min.
  Add 2ml of washing solution to the pellet and spin down at 750 rpm for 5 min.
  Add 6ml of MS medium + 0.6 M mannitol and incubate in dark for 24 hrs at RT (I usually leave it for 36 hrs).
  After 24hr-36hr, spin the protoplast at 750 rpm for 5 min.
  
• Incubate the protoplast pellet for at least 1 hr or O/N before RNA extraction.

Enzyme Solution ( 0.2 M KH 2 PO 4, 1 mM KNO3, 1mM MgSo4, 1μM KI, 0.01μM CuSo4. Add 0.6M mannitol and 10 mM CaCl 2. Adjust to pH 5.6)

Wash solution ( 0.6M mannitol and 10 mM CaCl 2 (pH 5.6)).

MS medium (4.4 g MS powder in1 litre of water. Add 0.6 M mannitol and 10 mM CaCl 2 (pH 5.6)).

2. Arabidopsis protoplast isolation and transfection

• Well expanded leaves were harvested from Arabidopsis plants of 3-4 weeks and surface sterilized for 10 min with 0.8% Clorox ? (sodium hypochloride), followed with 5 min washing with MilliRO water for three times.
• The leaves were sliced into 1 mm strips and incubated for 2-3 h at 25 oC in dark in enzyme mixture (0.4 M mannitol, 20 mM KCl, 20 mM MES, 1.5% cellulase R10 and 0.4% macerozyme R10 (Yakult Honsha, Japan), pH5.7, 10 mM CaCl 2 and 1% bovine serum albumin (BSA)). The enzyme solution was heated for 10 min at 55 degree and cooled to RT before CaCl2 and BSA were added.
• The protoplast suspension was then gently pumped for higher release efficiency and filtered through 70 um nylon screen.
• Filtrate was transferred into 50 ml sterile tubes and washed by centrifuge at 100 g for 2 min, and supernatant was discarded. Pellets were re-suspended in washing solution W5 (154 mM NaCl, 125 mM CaCl 2, 5 mM KCl, 2 mM MES, pH 5.7) and centrifuged with 100 g for 2 min.
• Protoplasts were transferred into 15 ml centrifuge tubes and centrifuged with 100 g for 2 min, and pellet was re-suspended in Culture Medium (0.4 M mannitol, 15 mM MgCl, 4 mM MES, 0.442% MS medium, pH 5.7) to a concentration of 4 X 10 4 protoplasts/ml. The protoplasts were kept on ice during all isolation steps and centrifuge was always conducted at 4 oC.
• Protoplasts of 200 μl were mixed well with 20 μg RNA transcripts and 200 μl polyethylene glycol (PEG) solution (40% PEG, 0.3 M mannitol, 100 mM CaCl 2), and 4.5 ml W5 solution was added to dilute the cells.
• The cells were kept on ice for 20 min before centrifuge with 100 g for 3 min. Supernatant was removed and the cells were washed again with 5 ml W5 solution when supernatant was removed completely to get rid of PEG.
• Cells were put in Culture Medium in Petri-dish at a concentration of 10 5 at 25 oC for incubation in dark.

3. Total RNA isolation by hot phenol method

• Grind sample leaves in liquid nitrogen into fine powder
• transferred to a 2 ml microfuge tube containing 1 ml hot phenol buffer {500 ml extraction buffer [ 0.1 M LiCl, 100 mM Tris-Cl (pH 8.0), 10 mM EDTA and 1% SDS] and 500 ml phenol, prewarmed at 80 °C
• vortex to mix well
• add 500 ul chloroform, vortex to mix well
• spin 12k×5 min, transfer the s/n to new tubes
• add equal volume of chloroform, vortex to mix well, spin 12k×5 min, transfer the s/n to new tubes
• add equal volume of 4M LiCl, mix well, precipitate the RNA at -20?C o/n
• spin 14k×20 min at 4?C, discard s/n, wash the pellet once with 2M LiCl
• re-suspend in 300 ul RNase-Free TE buffer;
• add 2.5 volume 100% ethanol and 0.1 volume 3M NaOAC pH 5.2, store at -20?C for 45 min to 1 hour(at least 20 mins)
• spin at 4?C 14k×20 min, discard s/n
• 70% ethanol (RNase- Free) 500 ul, spin 14k×10 min at 4?C, discard s/n, repeat once
• re-suspend in certain volume of RNase-Free water, check OD and stored at -80?C

4. Northern blot

Preparation of Probe RNA

• Linearize plasmid containing probe sequence
• phenol/chloroform purification of this linearized DNA, resuspend in RNase-Free water.
• In vitro transcription label RNA probe:
  
  

  Linearized template	(1ug) y ul
  10×DIG Labeling Mix	2 ul
  10×transciption buffer	2 ul
  T3/T7/SP6 RNA Polymerase	2 ul
  RNase-Free H2O	x ul

  
  Total volume 20 ul, mix well and incubate at 37?C for 2 hour.
• add 2 ul DNase I into the reaction mix, incubate at 37?C, 15 min, to remove the DNA template.
• purification of transcripts using 2V 100% ethanol and 0.1V NaOAC, on ice 10 min, spin 14k×20 min, discard s/n, wash the pellet twice with 70% ethanol (RNase Free), vacuum dry and re-suspend RNA in 20 ul or less RNase-Free water, stored at -80?C
  
  Separating RNA on Gel
• 0.2N NaOH solution soak the gel tank and combs for at least half an hour, then wash with RNase-Free water three times
• during the soaking, weigh 0.36g agarose in 30 ml 1×MOPS(10 ′ MOPS buffer [0.5 M MOPS, 0.01 M EDTA, pH 7.0]), dissolve in microwave oven and let cool to below 60?C, add 2 ml Formaldehyde
• pour gel and wait until polymerize, meantime, vacuum dry the RNA sample, then add 15-20 ul Sample Buffer([ 10 ′ MOPS buffer/formamide/formaldehyde/H 2O (1:1.8:5:2.2 )], incubate at 65?C for 10 min, chill on ice
• add 0.5-1 ul Loading Buffer for every sample
• pre-run the gel at 100 V for 10 min, then load the sample and run the gel at 50 V for about 2 hours
  
  Transfer and Blotting
• cut a gel sized membrane with marking, soak it in RNase-Free water for 10 min and then in 20×SSC(20×SSC: 3 M NaCl, 0.3 M trisodium citrate, Adjust pH to 7.0 (20°C) and autoclave).
  
• Soak the transfer plate in RO water and set up the apparatus
  Weigh 0.3 g agarose in 30 ml RO water, dissolve in microwave oven
  
• Place the membrane on the transfer plate, place the gel on the membrane, ( NO BUBBLE) seal with agarose gel, vacuum on
  
• Rinse with Solution 1,2 and 3 serially, at 55mBar,5 min each, discard. Solution 1(autoclaved ddH2O);Solution 2 (50mM NaOH, 10mM NaCl);Solution 3 (0.1M Tris.Cl pH7.4).
  
• Transfer with 20×SSC at 55mBar for 45 min, collect for reuse.
  transfer the membrane to a container with 5×SSC, wash for 5 min then dry the membrane on a C-Fold paper, cross-link.
  
• Stain the membrane in methylene blue solution (0.04% in 0.5M sodium acetate,pH5.2) for 5 min to check equal loading, wash the membrane in RNase-Free water, scan to save the file.

Blotting

• Put the membrane in 5 ml DIG Easy Hyb in a roller bottle, incubate at 68?C for 1 to 1.5 hour, meanwhile, pre-warm 5 ml DIG Easy Hyb at 68?C
  
• Withdraw the appropriate amount of labeled probe from the tube containing the product of the abeling reaction. Immediately add the denatured probe to a tube containing the appropriate amount of prewarmed DIG Easy Hyb (3.5 ml per 100 cm2) and mix by inversion to form the hybridization solution.
  
• Discard the pre-hybridization solution, put the 5 ml pre-warmed hybridization solution in the roller bottle, then add the probe into the roller bottle.Then incubate the membrane at 68?C o/n
  
• Prepare Low Stringency Buffer (2×SSC, 0.1% SDS) and High Stringency Buffer (0.1×SSC, 0.1% SDS, pre-warmed at 68?C)
  
• Transfer the membrane into a container with Low Stringency Buffer (2x SSC, containing 0.1% SDS), shaking for 5 min, discard, repeat once
  Add the pre-heated High Stringency Buffer (0.1x SSC, containing 0.1% SDS),shaking for 15 min, discard, repeat once at 68 degree.
  
• Add 10 ml Washing Buffer, shaking 2 min, discard,then add 10 ml Blocking Solution, shaking 30 min or longer, discard
  Add 10 ml Antibody Solution (for CSPD 1 to 10,000 dilution; for NBT/BCIP 1 to 5000 dilution), shaking 30 min, discard (this step can be incubate for longer time)
  
• Add 10 ml Washing Buffer, shaking 15 min, discard, repeat once
  
• Equilibrate membrane 3 min in 10 ml Detection Buffer, discard, meantime, prepare the detection solution: 10 ml Detection Buffer with 30 ul NBT (50 mg/ml) and 35 ul BCIP (50 mg/ml), add the detection solution, keep in DARK until clear bands come out
  
• Stop the reaction by washing the membrane in RO water.

5. Western blot

Sample Preparation
Use liquid nitrogen to grind sample leaves into find powder;
Add 1 ml Cracking Buffer for each sample and grind well;
Transfer into 1.5 ml tubes, 95?C boil for 5 min;
Spin at 10 g (about 10 krpm) for 10 min, transfer s/n to a new tube;
For every 15 ul s/n, add 1.5 ul 10×Loading Buffer or less.

Separate samples on SDS-PAGE

Make Resolving Gels and Stacking Gels according to the chart below

Resolving Gels

Stacking Gels (5%)

Buffers needed:

SDS-PAGE running buffer(5 times)(Glycine 94g pH8.3; 10%SDS 50ml; Tris base 15.1g)

While running the SDS-PAGE, prepare a gel-sized positive-charged Nylon membrane with proper markers, 4 pieces of Waterman Paper

Western Blot

Transfer from Gel to Membrane (keep it COOL)

• Wet the membrane in Methanol, and wet the Waterman Paper in Transfer Buffer;
• After the SDS electrophoresis, discard the stacking gel part;
• Set up the semi-dry transfer apparatus, NOTE: the transfer occurs from the gel (negative charged) to the membrane (positive charged), NO BUBBLES!
• Start transfer at 10V for around 1 hours
• Meanwhile, prepare 5% Blocking Buffer (2.5g milk powder in 50 ml TBST)

Transfer buffer:(Tris base 10mM 1.2124g/L; Glycine 96mM 7.206g/L; Methanol 100ml/L)

Blotting

• Transfer the membrane into a plastic container. Wash the membrane in 10ml TBST, shaking for 10 min. Repeat twice;
• Add Primary Antibody Solution 10 ul for 50 ml TBST Buffer, shaking for 1 hour;
• Wash the membrane in 10ml TBST, shaking for 10 min. Repeat twice;
• Add Secondary Antibody 5 ul for 50 ml TBST Buffer, shaking for 40 min;
• Wash the membrane in 10ml TBST for 10 min. Repeat twice;
• Add Detection Solution 10ml: AP Buffer 10ml+66 ul NBT (50 mg/ml)+33 ul BCIP (50 mg/ml), keep in dark;
• Stop the color development reaction by rinse the membrane with RO water, air-dry the membrane and store at RT.

Buffers used:

TBST: TBS + 0.05%Tween-20
TBS: (Tris base 1.2124g/L; NaCl 150mM 8.766g/L)
Alkaline phosphatase buffer:(Tris(pH9.5) 100mM 12.124g/L; NaCl 100mM 5.844g/L;MgCl2 5mM 1.165g/L)
Destaining solution:(30% methanol; 10% acetic acid; 60% H2O)


6. Simplified Arabidopsis transformation protocol
(Brief version for those who are familiar with the method)?

?? 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^oC 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" (9cm) pots.?
5. Before dipping, add Silwet L-77 to a concentration of 0.05% (500 ul/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 ug/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!?