Troubleshooting.

• All reagents in the kit should be stored according to the manual instructions.
• Before beginning the experiment, all components should be brought to room temperature.
• To ensure the uniformity and accuracy of each reagent, mix all components before use.
• Keep the temperature stable at room temperature throughout the entire experiment.
• The pH should be maintained between 7.2-7.4 for the entire duration of the experiment.
• Ensure all concentrated reagents are appropriately diluted prior to beginning the experiment.
• Washing technique should be kept consistent throughout the experiment and done according to the instructions in the manual.
• Avoid creating bubbles in solutions to ensure uniformity of all reactions.
• Take the reading of the microtiter plate 2 minutes after adding the stop solution.

Possible reasons:

• Degradation of the standard due to time or improper storage
• Standard curve dilutions were prepared incorrectly
• Pipetting errors leading to inconsistent concentrations
• Standard curve data was plotted improperly or without using appropriate software

Possible reasons:

• The reagents have expired - check packaging for expiration date
• Adding detection reagent A, B or TMB substrate was skipped
• The distilled water used for dilution was contaminated
• An enzyme inhibitor (such as Sodium azide) is present
• Wash buffer is too concentrated and has not been appropriately diluted
• The labelled enzyme is contaminated or inactive
• pH value of solution is too high or low. All solutions should be maintained at pH 7.2-7.4 for the entire experiment

Possible reasons:

• The reagents have expired or have not been stored in the correct conditions - check packaging for expiry date
• The reagents, standard, and/or sample were not warmed to room temperature prior to conducting the experiment
• Incorrect amounts and/or dilutions of reagents were used
• One or more solutions used were contaminated
• The labelled enzyme is contaminated or inactive
• TMB substrate was not incubated long enough
• Washing procedure problems, i.e.:
  • Wash buffer too concentrated
  • Wells were washed too many times
  • Wells were washed too vigorously
  • Wells were washed too long each time

Possible reasons:

• TMB substrate was stored incorrectly (not in a cool dark place and protected from light exposure)
• The incubation time was too long (can lead to strong, nonspecific adsorption)
• Wells were not washed adequately at one or more steps
• Cross contamination from pipette tips or other contaminated reagents
• Low concentration or low activation of the coated or detection antibody

Possible Reasons:

• Wells were washed inconsistently or insufficiently at one or more steps 
• Reagents were mixed or prepared inconsistently 
• One or more solutions were contaminated
• Bubbles formed in wells

Possible Reasons:

• Not enough sample was loaded in the well
• The amount of target protein is too low or is not expressed in the sample; consider using a positive control sample
• Poor transfer of target protein to membrane
• Antibody concentration is too low
• Antibody concentration is too high
• Not enough exposure time
• The substrate has been deactivated, or has not been incubated long enough
• Target protein was degraded or denatured during transfer; consider lowering the transfer temperature, decreasing the current or transfer time
• Membrane washing issues; consider reducing the frequency or duration of washing
• Antibody used is inactive or not concentrated enough

Possible Reasons:

• Experimental equipment is contaminated
• Membrane material is contributing to background
• Blocking buffer is interfering or cross-reacting with the antibodies
• Blocking step is not long enough
• Antibody concentration is too high
• Membrane washing issues; ensure there is no contamination and the washing step is long enough
• Exposure time is too long
• Other contamination during the experiment

Possible Reasons:

• Protein sample was digested during the experiment
• Too much sample was loaded in the well
• Blocking step is not long enough
• Washing step is not long enough
• Antibody concentration is too high
• Antibody specificity is too low
• The target protein used has multiple spliceosomes or modified sites

• Black spots on membrane – Blocking buffer is not suitable and may have non-specifically bound with the antibodies used
• White bands on membrane – Target protein or antibody concentrations are too high
• Molecular weight is not as expected – Gel is not mixed properly, or the concentration is incorrect
• Uneven or migrating bands – Equipment is not working properly; sample is not dissolved thoroughly; electrodes are not balanced; or too much sample was loaded

Possible Reasons:

• One or more steps have not been completed in the procedure
• Secondary antibody is not compatible with detection method used
• Primary and secondary antibody are not compatible
• Target protein is not expressed or not expressed enough in the sample
• Antibody concentration is not high enough
• One or both antibodies used has degraded
• Substrate has degraded
• pH of buffers used is not compatible with the experiment

Possible Reasons:

• Poor antigen retrieval; using heat-induced epitope retrieval and/or enzymatic digestion is recommended
• Too much liquid left on experimental area
• Area/section was not securely horizontal during incubation
• Proper fixation method was not used

Possible Reasons:

• Deparaffinization time was insufficient
• Endogenous enzymes/biotin were present
• Blocking time was not long enough
• Blocking buffer used was not a good match
• Antibody specificity was too low
• The washing step is not long enough
• Primary or secondary antibody concentration is too high
• DAB incubation time is too long
• Paraffin or cells have dried out
• Unwanted cross-reactivity with endogenous proteins and secondary antibody
• Antigen translocation due to sample treatment used

Possible Reasons:

• Target protein is not expressed or not expressed enough in the sample
• Antigen epitope was affected by immobilization
• Poor cell permeability
• Antigen was lost due to cell permeability; consider decreasing concentration or time of permeability reagent used
• Primary or secondary antibody concentration is too low
• Poor secondary antibody choice

Possible Reasons:

• Primary antibody is not specific enough
• Primary or secondary antibody concentration is too high
• Blocking time is insufficient
• IgG found in blocking buffer used
• Washing protocol is insufficient
• Cells have dried out
• Antigen was lost due to cell permeability; consider decreasing concentration or time of permeability reagent used
• Fluorescence microscope was not used with appropriate settings

Possible Reasons:

• The fluorescein-conjugated secondary antibody used has poor photo stability
• A sealing agent to prevent quenching was not used

Possible Reasons:

• Any auto-fluorescence was not quenched after fixing cells
• Some part of the sample exhibits auto-fluorescence; consider using a negative control and reducing background of the microscope
• Cell components present in the sample exhibit auto-fluorescence
• The amount of dead to living cells in the sample is too high

Possible Reasons:

• Improper storage or handling of antibodies used
• Antibody fluorescence was quenched before experiment
• cells used exhibit high auto-fluorescence similar to antibodies used
• Ineffective dye time or temperature
• Inappropriate procedure used for intracellular protein staining
• Attempted detection of extracellular secretory proteins
• Target protein has very low expression in sample used; consider using a brighter dye for better detection
• Antigens have been damaged by sample preparation or storage
• Improper functioning of flow cytometer laser; check calibration
• Inappropriate filters used for data collection
• Overcompensation of data
• Inappropriate cell gates used
• Poor data analysis

Possible Reasons:

• Cells used exhibit high auto-fluorescence
• Antibodies used have bound to dead cells; consider using active dyes
• Inappropriate use of Cy5 and other blue dyes
• Antibody concentration is too high
• Cells were dyed for too long
• Washing procedure was insufficient
• Experimental compensation regulation was insufficient

Possible Reasons:

• Incorrect concentration of isotype control used
• Poor match of isotype control and detection antibodies
• Adhesion or dead cells were included
• Experimental conditions have affected cell characteristics

Problem: Low DNA yield

• Possible Cause: incomplete sample lysis
• Possible Cause: Absolute ethanol not added to Buffer W2 before use
• Possible Cause: Ethanol not added to lysate before adding to column
• Possible Cause: pH of Buffer E is too low, or Buffer E has not been preheated
• Possible Cause: Poor quality starting material or sample was not stored appropriately

Problem: DNA has degraded

• Possible Cause: Sample was not fresh or stored appropriately, or was subjected to freeze-thaw cycles
• Possible Cause: DNase contamination

Problem: Inhibition of downstream enzymatic reactions

• Possible Cause: Purified DNA contains residual ethanol
• Possible Cause: Purified DNA contains residual salt

Problem: Low DNA yield

• Possible Cause: incomplete lysis
• Possible Cause: Ethanol not added to Buffer W2 before use
• Possible Cause: Incorrect elution conditions
• Possible Cause: Poor quality starting materials or inappropriate storage conditions

Problem: DNA has degraded

• Possible Cause: Sample is not fresh or has been subjected to repeated freeze/thaw cycles
• Possible cause: DNase contamination
• Possible Cause: Excessive pipetting or homogenization has damaged the DNA

Problem: RNA contamination

• Possible Cause: incomplete removal of RNA (use RNase treatment)

Problem: Inhibition of downstream enzymatic reactions

• Possible Cause: Purified DNA contains residual ethanol

Problem: Low DNA yield

• Possible Cause: Ethanol not added to Buffer W2 before use
• Possible Cause: Nuclease contamination
• Possible Cause: Column is overloaded; decrease the loading volume or starting sample amount
• Possible Cause: Gel has not completely dissolved; increase gel extraction time
• Possible Cause: incorrect elution conditions
• Possible Cause: Volume of elution buffer used is too small

Problem: Inhibition of downstream enzymatic reactions

• Possible Cause: Incorrect buffer used to elute DNA
• Possible Cause: Purified plasmid contains residual ethanol

Problem: DNA passes through in the flow-through or wash fraction

• Possible Cause: Column is overloaded; decrease the loading volume or starting sample amount
• Possible Cause: Inappropriate salt or pH conditions in buffers used

Problem: Purified DNA floats out of wells while running in agarose gels

• Possible Cause: Traces of ethanol not fully removed from column before eluting product

Problem: Low RNA yield

• Possible Cause: incomplete lysis or homogenization
• Possible Cause: Incorrect elution conditions

Problem: Low integrity or degraded RNA

• Possible Cause: RNase contamination

Problem: Inhibition of downstream enzymatic reactions

• Possible Cause: Purified RNA contains residual ethanol

Problem: Low yield

• Possible Cause: incomplete lysis
• Possible Cause: Residual buffer from previous steps present during the elution step
• Possible Cause: Viral nucleic acid stuck on column (repeat elution step and incubate the column for 5 min with water prior to centrifugation)

Problem: Degraded RNA

• Possible Cause: Poor starting material quality or inappropriate storage
• Possible Cause: RNase contamination

Poor RNA performance in downstream applications

• Possible Cause: purified product contains residual ethanol

Problem: DNA is sheared or degraded

• Possible Cause: lysate mixed too vigorously with pipette
• Possible Cause: DNase contamination
• Possible Cause: water (pH>7) present in elution/release step

Problem: RNA contamination

• Possible Cause: incomplete removal of RNA (use RNase treatment)

Problem: low DNA yields

• Possible Cause: Incomplete lysis and/or homogenizationPossible Cause: incorrect preparation of magnetic beads (vortex tube and fully resuspend beads before use)
• Possible Cause: Protein contamination (use Proteinase K treatment)
• Possible Cause: Incorrect elution/release conditions
• Possible Cause: poor starting material quality or inappropriate storage

Problem: High background on UV measurement

• Possible Cause: residual beads present in purified product (repeat magnetic separation if necessary)

Problem: RNA contamination

• Possible Cause: incomplete removal of RNA (use RNase treatment)

Problem: Plasmid bands smear on agarose gel

• Possible Cause: Plasmid DNA degradation (keep preparations on ice or frozen)

Problem: Poor plasmid quality

• Possible Cause: Genomic DNA contamination; do not overgrow bacterial cultures

Problem: Low DNA yield

• Possible Cause: Low plasmid copy number
• Possible Cause: Ethanol not added to Buffer W2 before use
• Possible Cause: Nuclease contamination
• Possible Cause: Column overloaded
• Possible Cause: the SDS in Buffer S2/M2 has precipitated in storage (to resolve, incubate at 30-40^oC and mix well)
• Possible Cause: Incorrect elution conditions
• Possible Cause: Plasmid lost in e. coli host

Problem: Inhibition of downstream enzymatic reactions

• Possible Cause: Incorrect buffer used to elute DNA
• Possible Cause: Purified plasmid contains residual ethanol

Problem: DNA passes through in the flow-through or wash fraction

• Possible Cause: Column is overloaded; decrease the loading volume or starting sample amount
• Possible Cause: Inappropriate salt or pH conditions in buffers used

Problem: Plasmid DNA floats out of wells while running in agarose gels

• Possible Cause: Traces of ethanol not fully removed from column before eluting product

Problem: Difficult to dissolve DNA

• Possible Cause: incomplete removal of ethanol
• Possible Cause: pellet has been overdried

Problem: RNA contamination

• Possible cause: incomplete removal of RNA (Use RNase treatment)

Problem: Low DNA yield

• Possible Cause: incomplete lysis and/or homogenization
• Suggestion: Grind tissue samples thoroughly, and ensure you are using the appropriate method for lysate preparation based on the amount of starting materials. Cut tissue samples into smaller pieces and ensure the tissue is completely immersed in lysis buffer
• Possible Cause: Presence of ethanol
• Possible Cause: separation phase was not repeated until interphase is clear
• Possible Cause: Incorrect Precipitation conditions or precipitation time was too short

Problem: DNA has degraded

• Possible Cause: Sample used was not fresh or was not stored appropriately
• Possible Cause: DNase contamination

Problem: Inhibition of downstream enzymatic reactions

• Possible Cause: Presence of ethanol in purified DNA

Problem: A260/280 ratio is below ~1.7

• Possible Cause: Incomplete removal of proteins or other contaminants

Problem: Difficult to dissolve RNA

• Possible Cause: incomplete removal of all ethanol

Problem: Genomic DNA contamination

• Possible Cause: incomplete removal of gDNA (use DNase treatment)

Problem: Degraded or low integrity RNA

• Possible Cause: RNase contamination (consider RNase inhibitor)

Problem: Low RNA yield

• Possible Cause: incomplete lysis and/or homogenization
• Suggestion: Grind tissue samples thoroughly, and ensure you are using the appropriate method for lysate preparation based on the amount of starting materials. Cut tissue samples into smaller pieces and ensure the tissue is completely immersed in lysis buffer
• Possible Cause: Incorrect precipitation conditions
• Possible Cause: Poor starting material quality or storage

Problem: Inhibition of downstream enzymatic reactions

• Possible Cause: Presence of ethanol in purified RNA