Collection and Storage of Cotton Leaf Tissue
Lyophilization of Cotton Leaves
- Harvest cotton tissue of interest. Separate the different leaf sample in a plastic
- Chill the samples on ice for transport back to the laboratory.
- Remove the midribs, if desired, then put the each sample in a 50-mL
polypropylene tube (PPT).
- Store the samples at –70°C until freeze-drying them.
- Freeze-dry (See Lyophilization below) the sample for 2 to 4 days.
- Store the freeze-dried leaf tissue in a 50-mL PPT tightly-capped at –20°C,
or in a desiccator at room temperature.
- Freeze the cotton leaf sample at –70°C in a deep freezer.
- Transport the sample in an ice chest.
- Check to see that the drain plug is closed (left side of condenser) and be
sure that the (break) switch is off and the ballast open.
- Turn on the bottom cool switch of the condenser.
- Turn on the top (pump, cool, control) switches of the chamber and set
“shelf temperature control” to –40°C or –50°C. Then, wait for the chamber
temperature to drop to the set level (it usually takes 1 to 3 hours).
- Turn on the bottom (pump) switch.
- Place the frozen samples on trays. Arrange in even layers. Placing a second
layer of tubes in the bottom 2 trays is OK. It may be fun to place the probes
into samples to watch the changing temperature.
- Close the chamber and wait for 1 to 2 minutes until the samples and
chamber temperature equalize. During this time, ice crystals will coat metal
parts of the chamber.
- Turn on the gauge switch, close the ballast, and wait for the vacuum to
reach <100 mT (about 5 to 10 min). In the meantime, condenser temperature
should be at least – 40°C.
- Set “shelf temperature control” to –20°C and let run at least 3 hrs, or
- Check the condenser periodically to be sure that the coil has not collected
enough ice to lug it up (rarely a problem on a fresh run).
- After the run is complete, turn off the (gauge) switch and open the ballast.
- Turn on (break) and turn off vacuum (pump) switches simultaneously.
- Remove the samples and turn off top (pump, cool, control) switches.
- Turn off (break) switch.
- Close the cap of samples tightly immediately.
- Place samples in a dry, airtight environment as soon as possible and store
out of direct sunlight.
Extraction Buffer (pH 6.0)
- Use fresh BEST or lyophilize the leaf tissue.
- Grind about 5 g of leaf tissue with mortar and pestle in liquid N2, transfer
the powder into a 50-mL polypropylene tube (50 mL), and store at –20°C.
- Add 25 mL of ice-cold extraction buffer, mix well, and put on ice until
centrifugation at 3750 rpm for 20 minutes (4°C); remove supernatant
(use a swinging bucket rotor).
- Add 10 mL lysis buffer and vortex to resuspend pellet; incubate in 65°C
water bath for 30 minutes. Mix the tubes periodically by stirring or
rocking the tubes every 10 minutes.
- Add 12 mL of chloroform:octanol (24:1) and mix gently, inverting the
tube, until an emulsion forms.
- Centrifuge at 3750 rpm for 20 minutes (15°C). Transfer the upper phase
to a new 50-mL tube through Miracloth.
- Add an equal volume of cold isopropanol, mix gently, and let it sit at
room temperature for more than 1 hour.
- Centrifuge at 2500 rpm for 10 minutes at room temperature. Decant
supernatant, and let tubes air dry. Then add 10 mL cold 76% ethanol/
0.2 M Na Acetate. Leave at 4°C for 1 hour or overnight. (Good stopping
- Centrifuge tubes (3750 rpm, 10 min). Carefully discard supernatant, and
rinse the pellet with cold 70% EtOH. Let tubes dry upside down on
paper towels (keep track of tube labels!).
- Let pellets dissolve into 3 mL TE buffer. Vortex at speed 5, and incubate
at 65°C for about 1 hour.
- Centrifuge at 3750 rpm for 10 minutes (15°C). Transfer supernatant to
a new 15-mL tube. Add RNAse to a concentration of 20 µg/mL, mix
gently, and incubate at 37°C for 15 minutes.
- Add 0.1 volume of 3M Na Acetate and 2 volumes of 95% ethanol for
DNA precipitation. Place at –20°C for 1–12 hours (overnight).
- Spool/scoop out the DNA on a 9" glass-hook pipette (new and unused)
and let it air dry under the hood or vacuum. If DNA cannot be spooled,
centrifuge at 2500 rpm for 10 min to form DNA into pellet. Once the
pellet is dry, place the pellet into 0.5 mL TE buffer in a 1.5-mL microfuge
tube for 30 minutes at 65°C.
- Determine both concentration and quality of the DNA with a spectrophotometer
with a 40 µL/800 µL dilution (1/20), and by running digested
and undigested DNA in a 1% agarose gel.
- 0.35 M glucose
- 0.1 M Tris HCl (pH 8.0)
- 5.0 mM Na-EDTA (pH 8.0)
- 2% PVP 40000 MW
- 0.1% DIECA, diethyldithiocarbamic acid (disodium salt)
- 0.2% beta-mercaptoethanol or Na2S2O5(add when used)
- 0.1 M Tris HCl (pH 8.0)
- 1.4 M NaCl
- 20 mM Na EDTA (pH 8.0)
- 2% CTAB
- 2% PVP 40000 MW
0.1% DIECA, diethyldithiocarbamic acid (disodium salt)
- 0.2% beta-mercaptoethanol or Na2S2O5 (add when used).