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We are dedicated to your success. Get back on track. View our expert recommendations for commonly encountered problem scenarios related to your protein-protein interaction experiments.
View the relevant questions below:
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Here are possible causes and solutions:
Cause | Solution |
Incorrect antibiotic used to select for transformants | Select for transformants on LB agar plates containing 10 μg/mL gentamicin (for bait plasmids) or 100 μg/mL ampicillin (for prey plasmids).
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Didn’t use the suggested LR Clonase™ II enzyme mix or LR Clonase™ II enzyme mix was inactive
| -Make sure to store the LR Clonase™ II enzyme mix at -20°C or -80°C. -Do not freeze/thaw the LR Clonase™ II enzyme mix more than 10 times. -Use the recommended amount of LR Clonase™ II enzyme mix -Test another aliquot of the LRClonase™ II enzyme mix.
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Not enough transformation mixture plated | Increase the amount of E. coli plated. |
Here are possible causes and solutions:
Cause | Solution |
Plates not replica cleaned | Replica clean immediately after replica plating, and again after 24 hours incubation.
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Inadequate replica cleaning | Review Appendix, page 85 of the manual. Immediately after replica cleaning, plate should contain no remaining visible cells.
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Too many cells transferred during replica plating | Review Appendix, page 85 of the manual. Transfer a minimal number of cells.
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Incorrectly prepared 3AT plates | Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.
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Incorrect incubation times | Incubate plates no longer than 60 hours (40-44 hours is usually best). Colonies arising after 60 hours are not likely to be of interest.
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Here are possible causes and solutions:
Cause | Solution |
Failure to add both bait and prey plasmids during transformation | Use bait and prey plasmids simultaneously in co-transformation procedures.
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Incorrect selection plates | Plate co-transformations on SC-Leu-Trp plates.
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Here are possible causes and solutions:
Cause | Solution |
Candidate clones were false positives | Candidate clones could have been mutants of bait that self-activate. See page 50 of the manual for additional information on false positives.
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Co-transformed pDEST™32 instead of bait plasmid | Retransform MaV203 with bait and prey plasmid.
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Multiple prey clones in the original 3ATR transformants | Examine more ampicillin E. coli transformants for additional prey clones.Test each by reintroduction.
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Here are possible causes and solutions:
Cause | Solution |
Incorrectly prepared 3AT plates | Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.
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Controls are too old or were mixed up | Return to the original DNA stocks provided, retransform on SC-Leu-Trp, and use fresh colonies.
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Uneven replica plating | When replica plating, maintain an even pressure across the entire surface of the master and selection plates. Uneven pressure can result in the failure of cells to transfer.
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Here are possible causes and solutions:
Cause | Solution |
Incorrectly prepared 3AT plates | Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.
|
Strains being tested do not contain bait and prey
| Confirm growth on SC-Leu-Trp plates. |
Uneven replica plating | When replica plating, maintain an even pressure across the entire surface of the master and selection plates. Uneven pressure can result in the failure of cells to transfer.
|
Cause | Solution |
Bait self-activates | Subclone segments of bait into pDEST™32 and retest.
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Incorrectly prepared 3AT plates | Review Appendix, page 75 of the manual. Confirm that all stock solutions were fresh and prepared correctly. Confirm that the calculation for amount of 3AT addition was correct.
|
Improper replica plating or replica cleaning | Review Appendix, page 85 of the manual. Immediately after replica cleaning, plate should contain no remaining visible cells (although a faint haze may be present on 3AT transformation plates).
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Incorrect incubation times | Incubate plates no longer than 60 hours (40-44 hours is usually best). Colonies arising after 60 hours are not likely to be of interest.
|
Here are possible causes and solutions:
Cause | Solution |
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.
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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.
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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.
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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.
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Here are possible causes and solutions:
Cause | Solution |
3AT concentration too low | Retest bait on various concentrations of 3AT.
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Plates made incorrectly | Review Recipes on page 75 of the manual.
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Improper replica cleaning | Review Appendix, page 85 of the manual. Immediately after replica cleaning, plate should contain no remaining visible cells (although a faint haze may be present on 3AT transformation plates).
|
Improper incubation times | Do not incubate plates longer than 60 hours. Colonies arising after 60 hours are not likely to be of interest.
|
Here are possible causes and solutions:
Cause | Solution |
E. coli not sufficiently competent | Use ElectroMAX™ DH10B™ cells for library.
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Too much DNA used | Use only 1 μl of DNA. Inhibitory compounds may reduce transformation efficiencies.
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Incorrect selection or concentration | Select for plasmid on LB+ampicillin (100 μg/mL).
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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.
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DNA suspended in incorrect buffer | Electroporation is sensitive to ionic strength. Suspend DNA pellet in TE.
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The protein-protein interaction may be transient in nature. Often times, adding crosslinkers to capture putative interacting partners inside or outside of the cell may maintain the interaction through lysis and enrichment/purification. Membrane permeable crosslinkers like DSS (Cat. No. 21658) allow one to “freeze” interactions inside the cell, whereas membrane impermeable crosslinkers like BS3 (Cat. No. 21585) only work on the cell surface or outside the cell.
There are several approaches to eliminate excess bands on a blot. These include the following:
Confirm that the reducing agent is fully cleaving the bait protein off of the target protein. Increase the concentration of the reducing agent or use fresh reducing agent if the bait:target protein is not being cleaved apart.
The following are the reasons and remedies for low signal in far-western blot application:
There are several approaches to eliminate excess bands on a blot. These include the following:
For Research Use Only. Not for use in diagnostic procedures.