What are the power supply considerations for the ZOOM™ IPGRunner system?

Product FAQ

Answer

The ZOOM™ IPGRunner™ System should be used with an external DC power supply designed for IEF and electrophoresis applications. This power supply must:

*Be isolated from the ground so that the DC output is floating.
*Be programmable, with a 4-protocol minimum.
*Be able to operate at low current (<1 mA) as IEF is performed at very low current.
Note: Many power supplies automatically shut off when the current drops below 1 mA. You will need a power supply capable of overriding the low current shut-off feature.

The electrical leads of the ZOOM™ IPGRunner™ Lid are recessed and may not fit into some power supply units. To allow connection of the ZOOM™ IPGRunner™ power leads with certain power supplies, use Invitrogen™ Power Supply Adapters available separately (Cat. No. ZA10001).

Answer Id: E10686

Was this answer helpful?

Yes
No
Thank you for your response

I ran an IEF gel and saw lines or bands across the whole gel upon staining it. Why is this?

Product FAQ

Answer

All lots of IEF gels that we manufacture exhibit these lines because carrier ampholytes do focus into tight bands extending across the gel and do stain a little. The intensity of these lines varies depending on the ampholytes used. One or more species may be overabundant, leading to more intense ampholyte bands. Usually these are still quite faint compared to the sample.

Answer Id: E10687

Was this answer helpful?

Yes
No
Thank you for your response

How can a solid yellow light on the Applied Biosystems™ 3730 Series DNA Analyzer be resolved?

Product FAQ

Answer

If a solid yellow light remains on the Applied Biosystems™ 3730 Series DNA Analyzer, perform the following steps:

1) Cycle power on the system in the proper order:
     a. Boot/Reboot the computer workstation and log in to the computer workstation
     b. Ensure that the buffer, water, and waste reservoirs are in their appropriate stations.
     c. Close the oven door, stacker drawer, and instrument doors.
     d. Cycle power on the Applied Biosystems™ 3730 Series DNA Analyzer.

2) Confirm that the original communication cable is attached properly ("computer" labeled end attached to computer and "instrument" labeled end attached to instrument). NOTE: the original communication cable must be used as it is of a non-standard design.

3) Reset the "3730User" account password. To do so:
     a. Right click on "My Computer" and click on "Manage".
     b. In the Computer Management window, click on "Local Users and Groups" and click on "User".
     c. On the right side of the window, there should be a username "3730User" Right click on that and select "Set Password".
     d. Set the password to "3730User" (case sensitive). Confirm the password entry then click on "Ok".
     e. In the Computer Management window, right click on "3730User" and select "Properties". "User cannot change password" and "Password never expires" should be selected.
     f. Click on "Ok" and exit out of Computer Management window.

4) Confirm whether or not your IT department has:
     a. Changed the name of the computer workstation.
     b. Installed or configured firewall/antiviral software on the computer workstation.
     c. Altered the original IP address of the computer/network card.
     d. Configured the computer for network access.
     e. Installed any updates or patches for the Operating System/Service Pack. NOTE: These changes may disrupt communication between the instrument and the computer.

5) Ensure that the "3730calib.ini" file is in the appropriate place. The file location on the Applied Biosystems™ 3730 Series DNA Analyzer running Data Collection v3.0 and v.3.1: C:/AppliedBiosystems/Firmware.

If the problems persist, contact Technical Support or Instrument Service.

Answer Id: E2546

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

Are there any suggestions on how to reduce high background when using the Colloidal Blue Staining Kit on Invitrogen™ Tris-Glycine gels?

Product FAQ

Answer

It may be possible to reduce background by using the protocol given for NuPAGE™ Invitrogen™ Bis-Tris/Small Peptides.

This protocol incorporates an extra fix step to remove excess SDS, which can act as an anti-colloidal agent and lead to higher background.

The low pH of the staining solution will fix the gel, but not as fast as the pre-fix step specified in the NuPAGE™ protocol.

Answer Id: E3714

Was this answer helpful?

Yes
No
Thank you for your response

Is DSP (Lomant's Reagent) water soluble?

Product FAQ

Answer

No, the reagent must first be reconstituted utilizing an organic solvent, such as dry DMSO or DMF. Note: It is recommended that the DMSO/organic solvent should not exceed 10% of the final reaction volume.

Answer Id: E13306

Was this answer helpful?

Yes
No
Thank you for your response

I saw bubbles appear in the later stages of the second hour of IEF gel electrophoresis. Can you please help me troubleshoot?

Product FAQ

Answer

The bubbles are probably caused by carbon dioxide outgassing from the gel. Carbon dioxide is a weak acid and will migrate into the acidic region of the IEF gel and come out of solution as it concentrates. Thorough degassing of the cathode buffer will reduce the problem. In addition, the gel can be run in the cold room so that the carbon dioxide won't come out of solution. If some bubbles form in the gel in the last few minutes of the run, they will probably not cause visible distortion. If they form earlier in the run, they may cause distortion because they act as insulators and change the gel temperature. These bubbles form between the gel surface and the cassette.

Answer Id: E10689

Was this answer helpful?

Yes
No
Thank you for your response

How often should the "Cleanup Processed Plates" command be run on the Applied Biosystems™ 3730 Series DNA Analyzer?

Product FAQ

Answer

The Applied Biosystems™ 3730 Series DNA Analyzer utilizes an Oracle™ database to store raw data generated by the system. Additionally, sample files extracted from the database are created on the hard drive. Routine maintenance must be performed to ensure that the appropriate database and hard drive space is available for new data; otherwise, new runs cannot be started and error messages will be generated. The "Cleanup Processed Plates" command in the Database Manager pane will allow you to delete all processed run data and processed plate records from the database. Before running this utility, ensure that all runs have been extracted from the database.

This utility does not delete:
- Data files that have already been extracted
- Spectral data and Spectral plate records
- Spatial information

Thermo Fisher Scientific recommends that this procedure be performed every 400–600 runs. If the database gets too full, an error message will advise the database cleanup. Extracted data (typically in the E drive) will also build-up over time and should be archived off of the computer workstation. After archiving, the extracted data should be deleted.

Answer Id: E2547

Was this answer helpful?

Yes
No
Thank you for your response

What shoud I do if R1, R2, R3, and/or R4 fails at Raw Traces and the screen turns red during the last step of initialization of my Ion PGM™ System?

Product FAQ

Answer

The pH of the nucleotides may be out of range or there may have been a minor problem during measurement. Press the 'Start' button and restart the measurement. If it passes, continue to start the run. If an error appears again, please note the pH of R1, R2, R3, R4, as well as the error message, and contact Technical Support.

Answer Id: EJP01165

Was this answer helpful?

Yes
No
Thank you for your response

What conditions are expected to remove Coomassie stain from proteins?

Product FAQ

Answer

The Coomassie dye binds to the protein via hydrophobic interactions, so anything that can disrupt these interactions can potentially serve to remove the dye.

For mild removal conditions, try nonionic detergents such as Triton X-100. If harsher conditions are acceptable, organic solvents like acetonitrile or alcohols should be effective.

Many times, washing with 30% ethanol will remove the stain. Changing the pH or salt concentration is not expected to help.

Answer Id: E3715

Was this answer helpful?

Yes
No
Thank you for your response

What is the recommended sample buffer and reconstitution buffer to obtain optimal cross-linking using DSP (Lomant's Reagent)?

Product FAQ

Answer

DSP is first dissolved in an organic solvent (such as dry DMSO) and added to an aqueous reaction mixture such as 20 mM sodium phosphate, 0.15 M NaCl, pH 7.5 (PBS). HEPES, bicarbonate/carbonate, and borate buffers may be substituted for PBS. Other buffers may also be substituted provided they do not contain primary amines. Recommended pH is 7-8. Once the cross-linker is reconstituted, use immediately.

Answer Id: E13307

Was this answer helpful?

Yes
No
Thank you for your response

How do you passage cells?

Product FAQ

Answer

The following is a suggested general procedure to rapidly remove various cell lines from the substratum while maintaining cellular integrity. This procedure is not meant to be universally applicable for all cell lines. The optimal conditions and concentrations employed for individual systems should be determined empirically. Cell viability should be routinely monitored at the time of subculturing. Cell viability should be greater than 90%.

1. Remove and discard spent cell culture media.

2. Wash cells using a balanced salt solution without calcium and magnesium or wash with EDTA. Add wash solution to the side of the flask opposite the cells. Rinse the cell sheet by rocking the flask for 1 to 2 min and discard wash solution.

3. Add the dissociation solution of choice at 2 to 3 ml/25 cm2 to the side of the flask opposite the cells. Be certain that the dissociation solution covers the cell sheet. Incubate the flasks at 37°C. Rock the flasks gently. Generally, cells are dissociated in 5 to 15 min. The actual time needed to dissociate cells will vary according to cell line. Monitor the process carefully to avoid cell damage. In addition to rocking gently, flasks of cell lines that are characteristically difficult to remove from the substratum may be tapped to expedite removal.

4. When the cells are completely detached, stand the flask in the upright position to allow the cells to drain to the bottom of the flask. Add complete media to the flask. Disperse the cells by pipetting repeatedly over the surface of the monolayer. Count and subculture the cells.

Reference:
Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.

Answer Id: E4297

Was this answer helpful?

Yes
No
Thank you for your response

Do you have a recommended single-step protocol for BP/LR recombination?

Product FAQ

Answer

Yes, we have come up with a single-step protocol for BP/LR Clonase™ reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.

Answer Id: E9857

Was this answer helpful?

Yes
No
Thank you for your response

After hybridization, how long can CytoScan&Trade; arrays be stored before washing and staining?

Product FAQ

Answer

Arrays must be put onto the fluidics station immediately after removal from the hybridization oven. Do not remove arrays from the oven you are ready to wash them.

Answer Id: E13985

Was this answer helpful?

Yes
No
Thank you for your response

What molar excess of Sulfo-NHS-LC-Biotin should I use to label antibodies and other proteins?

Product FAQ

Answer

The molar excess to use depends on the concentration of the ligand to be biotinylated. Although the literature contains references that use between 2-50 molar excess of biotin, we recommend the following: 10-fold molar excess for 10 mg/mL solution of protein; 25-fold molar excess for 2 mg/mL solution of protein.

Answer Id: E8562

Was this answer helpful?

Yes
No
Thank you for your response