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| - | == B-Galactosidase Activity Assay -- Marian Price-Carter, 9/7/00 ==
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| - | Modified From Protocols Online 10/15/10
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| - | (htt p://rothlab.ucdavis.ed u/proto cols/beta-galactosidase-3 .html)
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| - | Day 1: Start overnight lactose-free broth culture.
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| - | Negative control: cells lacking 13-galactasidase, such as Agrobacterium positive control: cells
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| - | with high enzyme activity E.coli.
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| - | Day 2: Dilute cells 1/100 in fresh medium now containing lactose, grow to mid-log.1 Prepare
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| - | solutions: Z buffer, phosphate buffer, ONPG2.
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| - | Preparation of Cells
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| - | Incubate cultures 20’ on ice to stop grnwth and wash:
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| - | ¯ Pellet at least 2 mL of cells at 4 C by centrifuging 10’ at 6,000 rpm in a Sorval SS34
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| - | rotor.
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| - | ¯ Pour off the supematant.
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| - | ¯ Resuspend the cell pellet in the same volume of chilled Z buffer.
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| - | ¯ Measure the ODr0o of the resuspended cells (blank against Z buffer)
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| - | Dilute cells in Z buffer to I mL (most easily done with a pippeter). For most activities, 0.5 mL
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| - | cells + 0.5 mL Z buffer will produce a desirable amount of yellow color in !-2 hours. For higher
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| - | levels (>500 Miller units), try 0. I mL cells + 0.9 mL Z buffer.
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| - | Permeabilize the diluted cells by adding 100 lal chloroform and 50 ~1 0.1% SDS (sodium
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| - | dodecyl sulfate, sodium laurel sulfate). Chloroform is easier to pippete if the air in the pippete tip
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| - | is saturated by dm~ving up and releasing chloroform several times.
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| - | Vortex; equilibrate the tubes 5’ in a 28 C water bath.
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| - | Assay
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| - | Start reaction by adding 0.2 mL substrate, o-nitrophenyl-13-D-galactoside (ONPG; 4 mg/mL)
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| - | ¯ Vortex - Record the time of addition precisely with timer or stopwatch.
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| - | ¯ Incubate the cells at 28 C.
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| - | ¯ Stop the reaction after sufficient yellow color has developed3 by adding 0.5 mL 1M
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| - | Na2CO34.
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| - | ¯ Vortex. - Note time of addition precisely.
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| - | ¯ Transfer 1 mL to an eppendorftube, spin 5’ at maximum to remove debris and
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| - | chloroform.
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| - | ¯ Record the optical density at 420 nm and at 550 nm for each tube. 5
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| - | ¯ Calculate the units of activity6"
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| - | This is basically the assay described by J.H. Miller in "Experiments in Molecular Genetics" 1972
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| - | Cold Spring Harbor Laboratories pages 352-355, with an extra step added. In the assay described
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| - | here, the cells are pelleted and resuspended in assay buffer (Z buffer) to eliminate error due to
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| - | the effects of different carbon sources in the growth medium on the [3-galactosidase enzyme
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| - | activity.
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| - | B-Galactosidase is able to hydrolyze (cleave) ~-D-galactosides. This enzyme facilitates growth
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| - | on carbon sources like lactose by cleaving it into a molecule of glucose and a molecule of
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| - | galactose which the cells can catabolize and grow on. In the assay described above, the substrate
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| - | o-nitrophenyl-I]-D-galactopyraniside (ONPG) is used in place of lactose. When the 13-
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| - | galactosidase cleaves ONPG, o-nitrophenol is released. This compound has a yellow color, and
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| - | absorbs 420 nm light. To measure ~-galactosidase activity the accumulation of yellow color
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| - | (increase’420 nm absorbance)/minute is monitored.
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| - | Footnotes
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| - | ~ In Sahnonella (which is naturally l]-galactosidase minus) this assay is used to monitor
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| - | transcription from insertion elements (that encode the [~-galactosidase enzyme) that have inserted
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| - | into different genes. The assay is usually performed on cells in the mid-log phase of growth. On
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| - | rich carbon sources like glucose, the OD6oo of a culture of wild-type Salmonella in mid-log phase
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| - | ranges from 0.28-0.7. On poorer carbon sources or in strains that have mutations in genes that
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| - | are important for groxvth, the OD6oo at mid-log phase may be lower, since the cells may enter
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| - | stationary phase at a lower density. Therefore, before doing the assay, it is important to follow
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| - | the growth of the strain of interest in each type of medium that will be used, plot a groxvth curve,
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| - | and determine when the cells are in mid-log phase in that particular medium.
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| - | Solutions for I]-galactosidase assays
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| - | Z buffer, per 50 mL:
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| - | ¯ O.80g Na2HPO.~.7H20 (0.06M)
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| - | ¯ 0.28g NaH2PO4.H20 (0.04M)
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| - | ¯ 0.5 mL 1M KC1 (0.01M)
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| - | ¯ 0.05 mL IM MgSO~ (0.001M)
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| - | ¯ 0.135 mL iB -mercaptoethanol (BME) (0.05M)
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| - | ¯ bring to approximately 40 mL with H20, dissolve all the salts
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| - | ¯ adjust the pH to 7.0
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| - | ¯ use a graduated cylinder to bring the buffer to 50 mL
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| - | ¯ store at4 C.
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| - | Note: BME is added to the reaction buffer to stabilize the ~-galactosidase enzyme. The important
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| - | part of BME is a reactive thiol (SH group). Thiols react with oxygen in the air and oxidize
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| - | (inactivate) over time. Therefore, try not to make much more Z buffer than you will use in a few
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| - | days. Store the unused portion at 4 C.
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| - | ONPG should be dissolved fresh each day. Dissolve 1.5X as much as you think you will need,
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| - | because you may have to repeat one or more of the assays Le. for a different amount of time or
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| - | with a different cell dilution. Dissolve the ONPG to a final concentration of 4mg/mL in 0. IM
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| - | phosphate buffer pH 7.0.
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| - | Phosphate buffer, per 100 mL:
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| - | ¯ 1.61g Na2HPO4.7H20 (0.06M)
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| - | ¯ 0.55g NaH2PO4.H~O (0.04M)
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| - | ¯ adjust the pH to 7.0
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| - | ¯ phosphate buffer is stable at room temperature and does not need to be made fresh each
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| - | time.
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| - | 3 What is sufficient yellow color? To get the most accurate measure of activity, the absorbance at
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| - | 420 nm (A42o) should range.from 0.6 to 0.9. Readings as low as 0.1 and as high as 1.2 are
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| - | acceptable. Tubes that have become as yellow as a tube of(unused) LB broth will probably be
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| - | sufficiently yellow.
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| - | If the reading is too low, try the assay again with more cells or longer incubation time. When the
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| - | element has inserted into a gene that is not expressed much, it will probably take hours to
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| - | develop enough yellow color. If your negative control starts to tum yellow (after several or more
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| - | hours) it means that the substrate is beginning to auto-hydrolyze. The assay can be left overnight.
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| - | The auto-hydrolysis is then accounted for by subtracting the A42o and A~oo of the negative control
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| - | from that of the tests before doing any further calculations.
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| - | If the reading is too high, try the assay again with fewer cells¯ Aim to stop the reaction after 15
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| - | minutes. For example, if in your first attempt, you added 0.5 mL 0fcells + 0.5 mL of Z buffer,
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| - | and it was too yellow after 5 minutes, try adding 0.1 mL cells + 0.9 mL of Z buffer. Watch the
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| - | tube carefully. Some cultures may have to be diluted even further[
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| - | ~ Adding the 1 M Na~CO3 stops the reaction by raising the pH of the solution to 11. At this pH
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| - | the enzyme is not active.
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| - | 5 The reading at 420 nm is a combination of absorbance by o-nitrophenol and light scattering by
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| - | cell debris. The absorbance at 550 corrects for light scattering. There is no absorbance from onitrophenol
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| - | at this wavelength. The light scattering at 420 nm is proportional to that at 550 rim:
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| - | light scattering at 420 nm = 1.75 x OD55o
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| - | 6 Use the following equation to calculate units of enzyme activity:
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| - | Miller Units = I000 x [(OD~o - 1.75 x OD~5o)] / (T x V x OD6~o)
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| - | ¯ OD~o and OD~o am mad from the reaction mixture.
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| - | ¯ OD6oo reflects cell density in the washed cell suspension.
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| - | ¯ T = time of the reaction in minutes¯
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| - | ¯ V = volume of culture used in the assay in mLs.
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| - | The units give the change in A4~o/min/mL of cells/OD6oo
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| - | Typical values:
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| - | a fully induced lac+ operon (+IPTG) = 1500 units
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| - | an uniduced lac÷ operon (no IPTG) = 1.5-3 units
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| | == 10/19/10 == | | == 10/19/10 == |
Today we are doing large scale DNA extractions from E-Coli Recovering t9002 and lac-z
Then placed the 4 tubes extracted from and placed them into centrefuge to spin down to extract DNA.
Then following protocol from pg 19. After extracting the DNA I placed into fridge in back Room @ 4C.
"
