scientificprotocols

Authors: Pat Heslop-Harrison and Trude Schwarzacher

REAGENTS

1. Metaphase arresting agents: Choose one of the following (Note 2) and shake vigorously to aerate before putting in living plant material; except for ice water, the solution should be the same temperature as that where the plants grow to avoid shock

• i. ice water (for cereals and temperate grasses): put distilled or deionized water in a clean plastic bottle, shake to aerate and keep at –20°C until the water starts to freeze, shake again.
• ii. 0.05% (w/v) colchicine (for most plant tissues). Can be stored in the dark at 4°C for several days to weeks
• iii. 2 mM 8-hydroxyquinoline (for dicotyledonous plants, particularly those with small chromosomes such as Arabidopsis thaliana ). Can be stored in the dark at 4°C for several days to weeks
• iv. a - bromonaphthalene : store water above liquid a -bromonaphthalene in a bottle (typically 100 ml over 50 ml). Shake, allow to separate, and then pipette out aliquots of the a -brom

scientificprotocols

Author: Pat Heslop-Harrison

Abstract:

Retroelements and their derivatives are an ubiquitous and abundant component of plant genomes. From the 1990s, PCR based techniques have been developed to isolate the elements from genomic DNA of different plants, and the methods and primers used are presented here.

Major classes of retroelements include the Ty1-copia, the Ty3-gypsy and the LINE (non-LTR) groups. Mixed PCR products representing the full heterogeneous pool of retrotransposons from each group are obtained by PCR with degenerate primers (degenerate nucleotide symbols link), as listed below. Some of the species tested are also listed, but experience of us and many others is that the primers succeed in most or all cases. Undegenerate primers for amplifying individual transposons are obtained from sequences in the databases. PCR protocols and programmes are given at the bottom of the page.

Revision 12/6/03: reduced amount of Taq polymerase recommended (we use enzyme from BioLine).

scientificprotocols

1. Freeze cotton leaves sample at -70 C in a deep freezer.

• Transport the sample in an ice chest box.
• Check to see that drain plug is closed (left side of condenser) and be sure that switch is off and ballast open.
• Turn on bottom switch of the condenser.
• Turn on top switches of chamber and set "shelf temperature control" to -400 or -50 C. Then, wait for chamber temperature to drop to set level (it usually takes 1 to 3 hr).
• Turn on bottom switch.
• Place the frozen samples on trays. Arrange in even layers. Placing second layer of tubes in 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 till the samples and chamber temperature equalize. During this time, ice crystals will coat metal parts of the chamber.
• Turn on switch and close ballast, and wait for vacuum to reach <100 mT (about 5 to 10 min). In the mean time, condenser temperature should be a

scientificprotocols

Author: David R. Caprette

Generation of a Light Curve

To address the hypothesis concerning photosynthetic efficiency it is necessary to expose sun and shade leaves to a range of light intensities long enough for them to fix significant amounts of carbon. It is necessary to expose identical surface areas under favorable conditions which are identical for all leaves except for light intensity (the experimental variable). A means of measuring the rate of carbon fixation is also necessary, of course.

Read over the recommended procedure for carbon-14 labeling of leaf disks, in which leaves are exposed to different light intensities in an atmosphere rich in the radioisotope carbon-14. Under favorable conditions, all newly-fixed carbon will include a proportion of labeled (radioactive) carbon. By measuring the amount of radioactivity in each disk, one can determine the amount of carbon fixed by each leaf.

After labeling the leaf disks,

scientificprotocols

Author: Saji Menon

Reagents and solutions

1. Alkaline Solution I

• 50 mM glucose
• 25 mM Tris HCl (pH 8.0)
• 10 mM EDTA (pH 8.0)
• 10 mg/ml lysozyme
• Alkaline Solution II

scientificprotocols

Author: Wei Ni

I use Leica TCS SP5 Confocal Microscope for imaging, red channel for DHE signaling (pay attention to the special excitation and emission parameters) and green channel for collagen auto-fluorescence (I used the default setting for Alexa 488). Here is an example of DHE staining on mouse aorta.

Procedure

1. Open image in Image J.

• Go to Image→Color→Split Channel. You will get three images in gray scale showing signals in red (image 1), green (image 2) or blue channel. I don’t have anything in blue channel, so just ignore the split result in blue channel. As you will see, I do have auto-fluorescent signal in the red channel, which was not showing obviously in the overlay. This is why I do not recommend only capture DHE signal and use the “measure” function, which will count both DHE signal and auto-fluorescent signal. (There is an exception. If you have super strong signal, you could use very low exposure time to avoid capturing th

scientificprotocols

Author: Gordon Laurie

1. Need six 150 mm dishes of cells. Remove from incubator, bring to bench, aspirate off medium, and wash each dish two times (15 ml each wash) with room temperature PBS. Place cells on ice between the washes.

• For later analysis of column eluate by avidin-peroxidase, cell surface proteins will need to be biotinylated. For sequence analysis, biotinylation can be omitted. To biotinylate, add 15 ml/dish of 50 µg/ml biotin (Pierce #21335 NHS-LC Biotin; prepare from freshly made stock [10 mg/ml in DMSO]). Incubate on ice for 90 min, aspirate biotin, then wash with (2) 5 ml/dish of PBS-glycine (0.56 gm glycine in 150 ml PBS).
• Remove cells by tapping the side of the dishes during the PBS-glycine washes if biotinylating, or in PBS wash if not biotinylating. Collect washes into two 50 ml conicals, spin for 5 min at 1500 rpm (4°C), aspirate supernatant, combine the cell pellets into one 50 ml conical, resuspend in 25 ml of PBS-glycine if biotinylated, or in 1 ml of lysis buffer on ic

scientificprotocols

Author: Daniel T. O'Connor

1. Can use 6- or 12-well tissue culture plates, although 6-well plates are probably better because the wells do not have to be 100% confluent to have plenty of cells per well to get good secretion counts (12- or 24-well plates are good for secretion studies using primary cultures).

• Consider Poly-L-Lysine or Poly-D-Lysine(Sigma) coating plates/wells if using a cell line that does not tightly adhere to the bottom (e.g., PC12 cells) and therefore may detach during the secretion study.
• Passage/seed cells onto plates to be used for secretion study 2 days before the study date for best results (we usually seed 2 days before, confluency 70-80%).
• For start of study: After removing old cell media, label cells with [3H]-L-norepinephrine ([3H]-NE). To label we add the[3H]-NE to pre-warmed (37oC) cell media to a final specific activity of 0.5-0.7 µCi/1 ml of media; we add 1 ml of[3H]-NE containing cell media to each well when using 6-well plates. Let the cells load in this media for

scientificprotocols

Author: Hancock Laboratory Methods

REFERENCE:

Hanock and Benz. 1986. BBA. 860:699-707.

PURPOSE:

Derivitization of amino groups to remove the positive charge characteristics of the protein, for example to determine the importance of amino groups to a protein's function.

scientificprotocols

Author: Sosnick Lab, University of Chicago

Description

For protein concentration, gas pressure is applied directly to ultrafiltration cell. Solutes above the membrane's molecular weight (MW) cut-off are retained in cell, while water and solutes below the cut-off pass into the filtrate and out of cell.

Membranes