Spectrophotometric Assay for GUS Activity
- To prepare 2 reagent blanks for the spectrophotometer, pipette 1 ml of buffer into each of 2 microfuge tubes. Label both tubes SB.
- To prepare an extract color blank for each extract (each putative transgenics and each control): pipet 900µl of buffer into one microfuge tube for each plant extract you have prepared (5). Label the 5 tubes with the plant number and a letter B for blank. Add 100 µl of the appropriate plant extract to each labeled tube of buffer and vortex gently to mix well. Make sure that you are using the correct reagent. You will have buffer, stop buffer, & assay reagent at your bench. DO NOT CONFUSE buffer with stop buffer!
- (You will perform your assays in duplicate.)For each plant extract to be assayed (5), pipet 900 µl of assay solution into each of two labeled microfuge tubes for a total of 10 tubes. (You may label both tubes the same-- just the plant number. Since they are replicates, they do not need to be distinguished from each other.) Add 100 µl of the appropriate plant extract to each of the two tubes of assay solution and vortex gently to mix well. Save the remainder of your extracts on ice.What is the effective concentration of the assay solution?
- Place all tubes (17) in the 37°C water bath and record the time. Prepare a set of 1.5ml plastic cuvettes for each of the 17 tubes with labels at the top of the cuvette.
- Incubate the reactions for exactly 2 hours. Stop the reactions by adding 400 µl of Stop Buffer (2.5 M 2-amino-2-methylpropanediol) to each tube (including all the blanks). Vortex well. What is the effective concentration of the stop buffer?
- Measure the A415 of each plant extract in a Hitachi double beam spectrophotometer. First zero according to the directions provided below using your 2 blanks labeled SB. Replace the front blank with first the extract color blank (#B). Record the absorbance and then replace the extract color blank with the first assay of that extract. Record the absorbance and then measure the absorbance of that plant's replicate assay. What does it mean if these values aren't identical? After you have recorded absorbances for all color blanks and all the enzyme activity assays, subtract the absorbance of each plant’s color blank from the plant’s activity absorbances. You should not get a negative number. If you do what does it mean? Speak with your instructor before continuing. Use those corrected absorbance values (if they are positive numbers) in the calculations below. Calculate the mean activity of the two assays for each plant and post the mean GUS activity to the course data spreadsheet in the lab. DO NOT LEAVE LAB WITHOUT POSTING ALL YOUR DATA!
HITACHI SPECTROPHOTOMETER OPERATION
TO READ ABSORBANCE (the machine requires at least 10 min. to warm up after turning it on before use):
MAIN MENU KEY
Press ENTER (2X)
Under Data Mode, select 1 for absorbance, ENTER
Test Set Up, ENTER
Sample num., select 1, ENTER
Wavelength, set desired wavelength (for GUS activity that would be 415nm), ENTER
Delay, set 1 or 2 seconds, ENTER
FORWARD KEY (wait for proper wavelength to appear on screen)
Place blank material in the reference and test positions, AUTO ZERO KEY
Remove blank material from test position and replace with sample, START KEY
Repeat for remaining samples, PAPER FEED KEY, take printed results or record manually in the table below and in your lab notebook.
50 mM NaPO4, pH 7.0
10 mM β-mercaptoethanol
10 mM EDTA
0.1% sodium lauryl sarcosine
0.1% Triton-X 100
Structural Evidence for Transgenic Plants
Buffer + 1.0% Pvpp (polyvinyl polypyrollidone)
Extraction Buffer with 1 mM PNPG (p-nitrophenyl β-D-glucuronide)
2.5 M 2-amino-2-methyl propanediol
Assaying the Transgenic Plants (HOME)
Leaf Extract Preparation
GUS Activity Assay by Histochemistry