Manual / Product Insert

Easy-DNA Isolation Kit

Version: 250056 MAN0000015 (1 October 2012)
Catalog #

Manual / Product Insert

ProQuest™ Two-Hybrid System

Version: 250871 REV A, 10/24/2005
Catalog #

Product Literature

Yeast and Fungal Testing

Product FAQ

I’m seeing a rapid pH shift in my medium. What should I do?

Answer

Please see the possible causes for this rapid pH shift, and suggested solutions:

- Incorrect carbon dioxide tension: Increase or decrease percentage of carbon dioxide in the incubator based on concentration of sodium bicarbonate in the medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use carbon dioxide amounts of 5-10%, respectively. Switch to carbon dioxide-independent medium.
- Overly tight caps on tissue culture flasks: Loosen caps one-quarter turn.
- Insufficient bicarbonate buffering: Add HEPES buffer to a final concentration of 10-25 mM.
- Incorrect salts in medium: Use an Earle’s salts-based medium in a CO2 environment and a Hanks’ salts-based medium in atmospheric conditions.
- Bacterial, yeast, or fungal contamination: Discard culture and medium. Try to decontaminate culture.

Answer Id: E11910

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Product FAQ

What cellular processes can be analyzed with a flow cytometer?

Answer

-Calcium flux: Each of the Oregon Green calcium indicators binds intracellular calcium with increasing affinity, providing a sensitivity range to match many applications. Oregon Green probes emit green fluorescence at resting levels of Ca2+ and increase their fluorescence intensity 14-fold with increasing Ca2+ concentration. The cell-permeant formulation (Cat. No. O6807) can be loaded in cell media and is compatible with flow cytometry.
-Rhodamine-based calcium indicators comprise a range of probes for large or small changes in Ca2+ concentration. They exhibit a 50-fold increase in fluorescence upon calcium binding and offer a range of wavelengths that can be used in conjunction with GFP or green-fluorescent dyes for multiplexing. Rhod-2, AM (Cat. No. R1245MP), in particular, localizes to mitochondria and can be used with flow cytometry.
-Membrane potential: A distinctive feature of the early stages of apoptosis is the disruption of the mitochondria, including changes in membrane and redox potential. We offer a range of products specifically designed to assay mitochondrial membrane potential in live cells by flow cytometry, with minimal disruption of cellular function. The MitoProbe family of mitochondrial stains (Cat. Nos. M34150, M34151, and M34152) provide quick, easy, and reliable flow cytometric detection of the loss of mitochondrial membrane potential that occurs during apoptosis. MitoTracker dyes (Cat. Nos. M7510 and M7512) are membrane potential-dependent probes for staining mitochondria in live cells. The staining pattern of MitoTracker dyes is retained throughout subsequent flow cytometry immunocytochemistry, DNA end labeling, in situ hybridization, or counterstaining steps. The Mitochondrial Permeability Transition Pore Assay (Cat. No. M34153) provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. The mitochondrial permeability transition pore (MPTP) is a non-specific channel formed by components from the inner and outer mitochondrial membranes, and appears to be involved in the release of mitochondrial components during cell death.
-Phagocytosis: In phagocytosis, cells internalize particulate matter such as microorganisms, and this process is important for immune responses and during the clearance of apoptotic cells. Probes for studying phagocytosis include BioParticles indicators—bacteria and yeast labeled with fluorescent dyes.
-Tracking phagocytosis using a quench/wash-based assay can report on simple uptake, or a pH indicator can be used to monitor stages in the pathway. We have no-wash assays labeled with pHrodo Red or Green (Cat. Nos. A10010, P35361, P35364, P35365, P35366, and P35367) and no-wash assays for whole blood (Cat. Nos. A10025, A10026, P35381, and P35382), all suitable for flow cytometry.
-pH changes: Sensitive pH determinations can be made in a physiological range using either fluorescent intensity or ratiometric measurements. pHrodo dyes (Cat. Nos. P35373 and P35372) provide signal intensity modulation from pH 2 to pH 9 and with a choice of fluorescent wavelengths. Tracking internalization of fluorescent dextran is a routine method for analyzing pH changes in cellular compartments. Dextran conjugates of pHrodo dyes (Cat. Nos. P35368 and P10361) provide the most complete solution by allowing discrimination of vesicles from early endosomes to lysosomes, with no quench or wash required.
-Reactive oxygen species: Cells that are environmentally stressed usually contain greatly increased levels of reactive oxygen species (ROS). CellROX reagents are fluorogenic probes developed for the detection and quantitation of ROS in live cells. These cell-permeant reagents are non-fluorescent or very weakly fluorescent in the reduced state; however, when oxidized, they become brightly fluorescent and remain localized within the cell. We offer CellROX Green (Cat. No. C10492), CellROX Orange (Cat. No. C10493), and CellROX Deep Red (Cat. No. C10491) Assay Kits validated for flow cytometry.

Answer Id: E14827

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Product FAQ

I performed a ProQuest Two-Hybrid forward screen and did not obtain any transformants (candidate interactors) on SC-Leu-Trp-His+3AT plates. Can you help me troubleshoot?

Answer

Here are possible causes and solutions:

- Gene of interest not in frame with GAL4 DNA Binding Domain encoding sequence: Sequence the DBD/test DNA junction.
- Poor quality cDNA library: Determine the percent of vectors containing inserts and their average size.
- Inadequate amount of cDNA library: Confirm concentration of library.
- Test DNA cloned into pDEST 32 lacks or masks a domain required for protein-protein interaction: Clone and test alternative segments of the test DNA (bait).
- cDNA library used does not contain proteins that interact with test protein X: Screen a cDNA library from an alternative tissue, developmental time point, or organism; Determine whether the bait protein is expressed in the library.
- Prey that interacts with bait may be toxic, unstable or require posttranslational modification: Some posttranslational modifications cannot be accomplished in yeast; Make sure a cDNA library is constructed in pDEST 22 or pEXP-AD502 and not in other high-copy-number AD-vectors.
- Bait may be toxic, unstable or require post-translational modification: Some posttranslational modifications cannot be accomplished in yeast; Subclone segments of bait into pDEST 32 and retest.

Answer Id: E15632

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Product FAQ

What is your recommended protocol for generating ss phagemid DNA?

Answer

The following protocol can be used to prepare ssDNA from DH12S or DH5aF', DH5aF'IQ, DH11S cells (this strain not currently offered for sale). Use a stock of the helper phage M13KO7 (1) that is of known titer. For convenience, DH12S cells are supplied with M13KO7 helper phage. DH12S cells are both endA positive, so single stranded DNA isolated from these strains tend to be less contaminated with double stranded rf DNA.

Small-Scale Preparation of ss Phagemid DNA:
1. Pick a single colony of cells containing phagemid DNA and resuspend in 2 mL TBG (1.2% tryptone, 2.4% yeast extract 0.4% glycerol, 17 mM KH2PO4 and 55 mM KH2PO4 and 20 mM glucose) containing 100 µg/mL ampicillin in a 15 mL tube.
2. Immediately add 10 µL M13KO7 helper phage stock at 10E11 pfu/mL.
3. Incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 2 hrs.
4. Add kanamycin to a final concentration of 75 µg/mL and incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 18 to 24 hrs.
5. Transfer 1.5 mL of culture to a sterile microcentrifuge tube and pellet cells by centrifuging at 14,000 x g for 10 min at 4 degrees C.
6. Transfer supernatant to fresh microcentrifuge tube and repeat the centrifugation.
7. Transfer 1.2 mL supernatant to a fresh microcentrifuge tube and add 300 µL of 2.5 M NaCl in 40% PEG 4000.
8. Vortex and incubate on ice for 15 min.
9. Centrifuge at 14,000 x g for 15 min at 4 degrees C.
10. Resuspend the pellet in 50 µL TE and phenol extract to remove the viral coat.
11. Use 10 µL of the final 50 µL volume for gel analysis.
This protocol yields 0.5 to 1 µg ss phagemid DNA.

Large-Scale Preparation of ss Phagemid DNA:
1. Resuspend a single colony in 5 mL of TB or LB broth containing 100 µg/mL ampicillin in a 15-mL tube.
2. Shake at 37 degrees C and 275 rpm overnight.
3. Add 100 µL of the overnight culture to 200 mL LB broth and 100 µg/mL ampicillin in a 1 L flask. Incubate at 37 degrees C with shaking (275 rpm) for 3 hrs.
4. Add 200 µL of M13KO7 helper phage (1 x 10E11 pfu/mL) to the culture and continue to incubate for 2 hrs.
5. Add 1.5 mL of 1% (w/v) kanamycin to the cells for a final concentration of 75 µg/mL. Incubate the infected cells for an additional 18 to 24 h at 37 degrees C.
6. Centrifuge this culture at 16,000 x g for 15 min at 4 degrees C .
7. Filter the supernatant through a 0.2 µm sterile filter into an autoclaved centrifuge bottle. Add 40 µL of DNase I (50 units/µL) and incubate at room temperature for 3 hrs. This step should remove any residual ds plasmid DNA contamination.
8. Transfer 100 mL of the supernatant to another centrifuge bottle. Add 25 mL of 2.5 M NaCl in 40% PEG 4000 to each of the centrifuge bottles containing the supernatant.
9. Vortex the mixture, incubate on ice for 1 h, and centrifuge at 16,000 x g for 20 min at 4 degrees C.
10. Carefully discard the supernatant. To fully drain off the remaining solution from the pellets, place the bottles on an angle, with the pellet side facing up for 10 to 15 min. Remove the solution with a sterile Pasteur pipet.
11. Resuspend the pellets in 2 mL of TE buffer. Add 10 µL of proteinase K solution (20 mg/mL), 20 µL of 10% SDS, and incubate this mixture at 45 degrees C for 1 hr.
12. Transfer the digested mixture to three microcentrifuge tubes and extract four times with an equal volume of phenol:chloroform: isoamyl alcohol (25:24:1), precipitate with ethanol, and dissolve in 100 µL TE buffer.
13. Freeze the solubilized DNA at -20 degrees C for 1 hr and centrifuge in a microcentrifuge at 14,000 x g for 15 min at 4 degrees C.
14. Transfer the supernatant containing the ss plasmid DNA to a fresh tube and discard the polysaccharide pellet. Store the ssDNA at 4 degrees C.
15. Determine the DNA concentration (OD260).
This protocol yields ~100 to 200 µg ss phagemid DNA.

(1) Vieira, J. and Messing, J. (1987) Methods in Enzymology 153, 3.

Answer Id: E4161

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Product FAQ

What is the recommended protocol for preparation of competent yeast for large-scale transformation with bait and plasmid-library in the ProQuest system?

Answer

(1) Suspend several colonies of MaV203 in 50 µL autoclaved, distilled water in a microcentrifuge tube and spread it onto the center of a 10-cm YPAD plate using an autoclaved loop or toothpick. Repeat procedure for a second YPAD plate. Incubate both plates for 18-24 h at 30 degrees C. 

(2) Scrape and completely suspend the cells (by brief vortexing and pipetting up and down) in 10 mL autoclaved, distilled water. Add a sufficient volume of this cell suspension to two 1-L flasks each containing 500 mL liquid YPAD medium to give an OD600 of ~0.1. Reserve approximately 10 mL YPAD medium to use as a blank in the spectrophotometer. 
Note: Perform serial 1:10 dilutions in water of the 10-mL cell suspension then determine the OD600 of each dilution to allow an estimate of cell suspension required to produce the desired OD of 0.1. Appropriate cell densities require that the measured OD be <1.0. Verify that the OD is ~0.1 after inoculation. Use plastic cuvettes for all OD600 measurements. 

(3) Shake (~250 rpm) at 30 degrees C until the OD600 reaches ~0.4 (usually ~5 h). Read the OD. 

(4) Prepare fresh: 
225 mL 1X TE/LiAc by combining 22.5 mL 10X TE, 22.5 10X LiAc, and 180 mL autoclaved H2O. 
30 mL PEG/LiAc by combining 3 mL 10X TE, 3 mL 10X LiAc, and 24 mL 50% PEG-3350. 
200 µL carrier DNA by boiling sonicated herring sperm DNA or sonicated salmon sperm DNA (10 mg/mL) for 5 min and placing on ice until use. 

(5) Split each 500 mL of yeast cells into two conical 250-mL tubes and centrifuge at 3,000 x g for 5 min at room temperature. 

(6) Pour off the supernatants and gently suspend each pellet by pipetting up and down in 100 mL autoclaved, distilled water at room temperature. 

(7) Centrifuge at 3,000 x g for 5 min at room temperature. Pour the supernatant off of the centrifuged cells and suspend each cell pellet in 50 mL 1X TE/LiAc solution. 

(8) Centrifuge at 3,000 x g for 5 min at room temperature. Carefully pour off the supernatants and suspend each cell pellet in a final volume of 1 mL 1X TE/LiAc solution and pool all suspensions for a total of 4 mL. 

(9) Perform 30 transformations. Combine 4 mL of cells, 200 µL freshly boiled carrier DNA and 150 µg (~1 µg/µL) bait plasmid DNA and 150 µg (~1 µg/µL) plasmid-library plasmid DNA. Mix gently by pipetting up and down. Add 24 mL PEG/LiAc solution and mix gently, but completely. Aliquot into 30 autoclaved microcentrifuge tubes of 950 µL each.

(10) Incubate for 30 min in a 30 degrees C water bath. 

(11) Heat shock for 15 min in a 42 degrees C water bath. 

(12) Centrifuge in a microcentrifuge (6,000 - 8,000 x g) for 20-30 s at room temperature. Carefully remove the supernatant. 

(13) Gently suspend each pellet in 400 µL autoclaved, distilled water by pipetting up and down. 

(14) To estimate the total number of transformants, plate two dilutions of the transformation. Mix 10 µL of transformation with 90 µL autoclaved, distilled water. Plate 100 µL on a 10-cm SC-Leu-Trp plate (1:800 final dilution factor). Mix 10 µL of transformation with 990 µL autoclaved, distilled water. Plate 100 µL on a 10-cm SC-Leu Trp plate (1:8,000 final dilution factor).

Answer Id: E15610

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Product FAQ

What is the formulation of the SOC medium that is provided with competent cells?

Answer

SOC (Super Optimal Catabolite) Medium Preparation (for 1 Liter):

1) To a 2 Liter flask with stir bar add the following:
- Bacto Tryptone 20 g
- Yeast Extract 5 g
- Sodium Chloride (NaCl) 0.58 g
- Potassium Chloride (KCl) 0.186 g
2) Add sterile water to a final volume of 1 Liter.
3) Mix well on magnetic stir plate for 5-10 minutes or until all of the ingredients are well mixed and completely dissolved.
4) Autoclave 30 minutes.
5) Allow to cool to room temperature.
6) Add 10 ml of sterile 2M Magnesium Solution (1M Magnesium sulfate, 1M Magnesium chloride)and mix well.
7) Add 10 ml of sterile 2M Glucose and mix well. (Final Glucose concentration is 20 mM).

Answer Id: E3343

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Product FAQ

How do I prepare lawn cells for M13 cloning?

Answer

Lawn cells for M13 infection must be made with an E. coli strain containing the F' episome. Some of our competent cells that may be used include: DH5aF'IQ, INValphaF', TOP10F', OmniMAX2, Stbl4, or DH12S cells.

To make a glycerol stock of lawn cells:
1) Streak cells on an LB plate and incubate overnight at 37C.
2) Pick a single colony from the plate into 50 ml LB or S.O.B. medium and incubate the culture at 37C until the OD550 reaches 2.0.
3) Centrifuge the cells in a clinical centrifuge at 3,000 x g for 10 min at 4C.
4) Resuspend the pellet in 5 ml 60% S.O.B. and 40% glycerol.
5) Aliquot the cells and freeze them in a dry ice/ethanol bath. Store the cells at -70C.

LB Medium (per liter):
- 10 g Tryptone (SELECT Peptone 140)
- 5 g Yeast Extract (SELECT Yeast Extract)
- 10 g NaCl
Add Distilled Water to a final volume of 1 L. Stir to dissolve, and autoclave.

S.O.B. Medium (per liter):
- 20 g Tryptone (SELECT Peptone 140)
- 5 g Yeast Extract (SELECT Yeast Extract)
- 10 ml 1 M NaCl - 2.5 ml 1 M KCl - 10 ml 1 M MgCl2 - 10 ml 1 M MgSO4
Add tryptone, yeast extract, NaCl, and KCl to a flask or bottle, and add Distilled Water to a final volume of 1 L (~980 ml). Stir to dissolve, autoclave, and cool to room temperature. Then add Mg++ stock solutions (1 M MgCl2/6H2O and 1 M MgSO4/7H2O, filter sterilized). Mg++ solutions should not be added before autoclaving, as this will likely result in precipitation.

Answer Id: E4160

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Product FAQ

I am using your ProQuest Two-Hybrid System and am at the step of isolating the prey plasmid. I am not getting enough E. coli transformants. What should I do?

Answer

Here are possible causes and solutions:

- E. coli not sufficiently competent: Use ElectroMAX DH10B cells for library.
- Too much DNA used: Use only 1 µl of DNA. Inhibitory compounds may reduce transformation efficiencies.
- Incorrect selection or concentration: Select for plasmid on LB+ampicillin (100 µg/mL).
- Alternative yeast DNA preparation procedure used: Use the method described on page 45 of the manual. Other procedures designed for high-copy-number vectors may not work with the ARS/CEN based vectors used here.
- DNA suspended in incorrect buffer: Electroporation is sensitive to ionic strength. Suspend DNA pellet in TE.

Answer Id: E15634

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Product FAQ

What are some common methods for analyzing protein-protein interactions?

Answer

Here are some common methods for analyzing protein-protein interactions:

- Co-immunoprecipitation (Co-IP)
- Pulldown assays
- Yeast two-hybrid screening
- Crosslinking
- Label transfer
- Immunofluorescence/fluorescence resonance energy transfer (FRET)
- Far-western analysis
- Surface plasmon resonance: relates binding information to small changes in refractive indices of laser light reflected from gold surfaces to which a bait protein has been attached. Changes are proportional to the extent of binding. Special labels and sample purification are not necessary, and analysis occurs in real time.
- NMR (nuclear magnetic resonance): Method that can provide insights into the dynamic interaction of proteins in solution.
- Mass Spectrometry: Used in concert with affinity-based methods (such as co-IPs) to isolate binding partners and complexes and to identify the component proteins using standard mass spectral methods (e.g., MALDI-TOF and mass searching of bioinformatics databases). Heavy (deuterated) crosslinkers can be used in conjunction with mass spectroscopy to elucidate details about protein-protein interactions.
- X-ray crystallography: crystallization of the interacting complex allows definition of the interaction structure.

Answer Id: E15599

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Product FAQ

What is the recommended protocol for preparation of competent yeast for large-scale transformation with bait and plasmid-library in the ProQuest system?

Answer

(1) Suspend several colonies of MaV203 in 50 µL autoclaved, distilled water in a microcentrifuge tube and spread it onto the center of a 10-cm YPAD plate using an autoclaved loop or toothpick. Repeat procedure for a second YPAD plate. Incubate both plates for 18-24 h at 30 degrees C.

(2) Scrape and completely suspend the cells (by brief vortexing and pipetting up and down) in 10 mL autoclaved, distilled water. Add a sufficient volume of this cell suspension to two 1-L flasks each containing 500 mL liquid YPAD medium to give an OD600 of ~0.1. Reserve approximately 10 mL YPAD medium to use as a blank in the spectrophotometer.
Note: Perform serial 1:10 dilutions in water of the 10-mL cell suspension then determine the OD600 of each dilution to allow an estimate of cell suspension required to produce the desired OD of 0.1. Appropriate cell densities require that the measured OD be <1.0. Verify that the OD is ~0.1 after inoculation. Use plastic cuvettes for all OD600 measurements.

(3) Shake (~250 rpm) at 30 degrees C until the OD600 reaches ~0.4 (usually ~5 h). Read the OD.

(4) Prepare fresh:
225 mL 1X TE/LiAc by combining 22.5 mL 10X TE, 22.5 10X LiAc, and 180 mL autoclaved H2O.
30 mL PEG/LiAc by combining 3 mL 10X TE, 3 mL 10X LiAc, and 24 mL 50% PEG-3350.
200 µL carrier DNA by boiling sonicated herring sperm DNA or sonicated salmon sperm DNA (10 mg/mL) for 5 min and placing on ice until use.

(5) Split each 500 mL of yeast cells into two conical 250-mL tubes and centrifuge at 3,000 x g for 5 min at room temperature.

(6) Pour off the supernatants and gently suspend each pellet by pipetting up and down in 100 mL autoclaved, distilled water at room temperature.

(7) Centrifuge at 3,000 x g for 5 min at room temperature. Pour off the supernatant of the centrifuged cells and suspend each cell pellet in 50 mL 1X TE/LiAc solution.

(8) Centrifuge at 3,000 x g for 5 min at room temperature. Carefully pour off the supernatants and suspend each cell pellet in a final volume of 1 mL 1X TE/LiAc solution and pool all suspensions for a total of 4 mL.

(9) Perform 30 transformations. Combine 4 mL of cells, 200 µL freshly boiled carrier DNA and 150 µg (~1 µg/µL) bait plasmid DNA and 150 µg (~1 µg/µL) plasmid-library plasmid DNA. Mix gently by pipetting up and down. Add 24 mL PEG/LiAc solution and mix gently, but completely. Aliquot into 30 autoclaved microcentrifuge tubes of 950 µL each.

(10) Incubate for 30 min in a 30 degrees C water bath.

(11) Heat shock for 15 min in a 42 degrees C water bath.

(12) Centrifuge in a microcentrifuge (6,000 - 8,000 x g) for 20-30 s at room temperature. Carefully remove the supernatant.

(13) Gently suspend each pellet in 400 µL autoclaved, distilled water by pipetting up and down.

(14) To estimate the total number of transformants, plate two dilutions of the transformation. Mix 10 µL of transformation with 90 µL autoclaved, distilled water. Plate 100 µL on a 10-cm SC-Leu-Trp plate (1:800 final dilution factor). Mix 10 µL of transformation with 990 µL autoclaved, distilled water. Plate 100 µL on a 10-cm SC-Leu Trp plate (1:8,000 final dilution factor).

Answer Id: EF

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Manual / Product Insert

Protocol: Galacto-Star™ System (English )

Version: 20 Nov 2008
Catalog #
  • T1012
  • T1014
  • T1056
  • T1013(Discontinued)
  • T1019(Discontinued)
  • T1020(Discontinued)
  • T1021(Discontinued)

Manual / Product Insert

InsectSelect Glow System with pIZT/V5-His

Version: Version H 10/20/10