- ELISA Troubleshooting Guide:
- What are the common problems occurred in bacteria culturing? How to solve them?
- What are the common problems encountered in protein gel electrophoresis? What are the causes and solutions?
- What are the common problems encountered in cell culturing? What are the causes of the problems? How to solve them?
- What are the common DNA sequencing problems encountered? What are the causes of the problems? How to solve them?
- Can I reuse DNA samples which have been measured using spectrophotometer for downstream processing?
- Why do I get low efficiency of DNA amplification when I have used filter tips for preparation of PCR experiments?
- Why the transfection efficiency is low for my cell lines or RNAi by using plasmid as vector?
- What is PCR grade primer, HAP purified primer, PAGE and HPLC purified primer?
- What are the precautions in RNA extraction?
- Why my ligation fail?
Problem 1: Signal is high, stan-dard curves have saturated O.D.’s
Probable Cause Solution Standard reconstituted with less volume than required Reconstitute lyophilized standard with correct volume of solution recommended in the protocol. Plate incubation was too long Decrease incubation time. Detection antibody incuba¬tion time is too long Decrease detection antibody incubation time. Avidin-HRP incubation time is too long. Decrease Avidin-HRP incubation time. Substrate solution incuba¬tion time is too long Decrease substrate solution incubation time.
Problem 2: Sample readings are out of range
Probable Cause Solution Samples contain no or below detectable levels of analyte If samples are below detectable levels, it may be possible to use higher sample volume. Check with technical support for appropriate protocol modifications. Samples contain analyte concentrations greater than highest standard point. Samples may require dilution and reanalysis.
Problem 3: High variation in samples and/or standards
Probable Cause Solution Multichannel pipette errors Calibrate the pipettes. Plate washing was not adequate or uniform Make sure pipette tips are tightly secured. Confirm all reagents are removed completely in all wash steps. Non-homogenous samples Thoroughly mix samples before pipetting. Samples may have high particulate matter Remove the particulate matter by centrifugation. Insufficient plate agitation The plate should be agitated during all incubation steps using an ELISA plate shaker at a speed where solutions in wells are within constant motion without splashing. Cross-well contamination When reusing plate sealers check that no reagent has touched the sealer. Care should be taken when using the same pipette tips used for reagent additions. Ensure that pipette tips do not touch the reagents on the plate.
Problem 4: Background is high
Probable Cause Solution Background wells were contaminated Avoid cross-well contamination by using the sealer appropriately. Use multichannel pipettes without touching the reagents on the plate. Matrix used has en¬dogenous analyte or interference Check the matrix ingredients for cross reacting components (e.g. interleukin modified tissue culture medium). Insufficient washes Increase number of washes. Increase soaking time between washes prior to addition of substrate solution. TMB Substrate Solution was contaminated TMB Substrate Solution should be clear and colorless prior to addition to wells. Use a clean container prior to pipetting substrate solution into wells.
Problem 5: No signal
Probable Cause Solution Incorrect or no Detection Antibody was added Add appropriate Detection Antibody and continue. Avidin-HRP was not added. Add Avidin-HRP according to protocol and continue. Substrate solution was not added. Add substrate solution and continue. Wash buffer contains sodium azide Avoid sodium azide in the Wash Buffer.
Problem 6: Low or poor signal for the standard curve
Probable Cause Solution Standard was incompletely reconstituted or was inappropriately stored Reconstitute standard according to protocol. Store reconstituted standard in appropriate vials. Store reconstituted standard at -70°C. Reagents added to wells with incorrect concentra¬tions Check for pipetting errors and correct reagent volume. Incubations done at inappropriate temperature, timing or agitation Assay conditions need to be checked.
Problem 7: Colour developing slowly
Probable Cause Solution Plates are not at the correct temperature Ensure plates are at room temperature and that the reagents are at room temperature before use Conjugate too weak Prepare the substrate solutions immediately before use. Ensure the stock solutions are in date and have been stored correctly, and are being used at the correct concentration. Ensure the reagents are used as directed, at the correct concentration. Contamination of solutions Presence of contaminants, such as sodium azide and peroxidise can affect the substrate reaction. Avoid using reagents containing these preservatives.
Problem 8: Edge Effects
Probable Cause Solution Uneven temperatures around work surface Avoid incubating plates in areas where environmental conditions vary. Use plate sealers
Problem 9: Drift
Probable Cause Solution Interrupted assay set-up Assay set-up should be continuous - have all standards and samples prepared appropriately before commencement of the assay Reagents not at room temperature Ensure that all reagents are at room temperature before pipetting into the wells unless otherwise instructed in the antibody inserts
Problem 1: No growth after 24 hours overnight culturing
- Source culture was dead.
- Correct medium or growth conditions for the strain was not provided.
- Transfer broth cultures once a week.
- Transfer plate or slant cultures once a month.
- Grow cultures only until there is healthy growth, usually overnight.
- Do not keep plates over a weekend in an incubator.
- Refrigerate cultures after there is growth, do not let them grow too long.
- If the culture used as an inoculum was young, refer to problem 2.
Problem 2: The culture is growing very slowly
- Optimum growth conditions was not provided, growth requirements of the bacteria are not met.
- Fungi basically grow slowly (takes 3-5 days to grow)
- Determine the optimum growth conditions for the strain and provide it as best as possible.
- Most bacteria grow aerobically, so growing cultures in flasks with a large surface area: volume ratio will help to provide oxygen.
- Shaking is desirable but not usually necessary.
- Yeast grows better in a growth medium that contains sugar. Baker’s yeast grows fine in sugar and water.
- Most bacteria and fungi grow slowly at room temperature. An incubator can help to speed up growth.
Problem 3: Bacterial cells on plates or broth disappeared
- Some strains, notably those of the genus Bacillus, destroy their cells when they die. In this case you can tell when a culture is dead because the cells lyse and are no longer visible.
- If the problem occurs with a Bacillus strain, you may be able to rescue the culture by transferring some of the invisible cell material to fresh broth. Many strains of Bacillus produce resistant spores before the vegetative cells lyse and it requires only a few spores to start a new culture.
- Obtain a fresh culture if the strain cannot be rescued.
Problem 4: The culture plate has colonies that do not look like most of the colonies, or there are colonies where nothing was streaked.
- The plate was contaminated with bacteria or fungi.
- If there is little contamination and it is clear which colony type is the desired one, restreak from single colony.
- Alternatively, obtain a fresh strain.
3) What are the common problems encountered in protein gel electrophoresis? What are the causes and solutions?
Problem 1: Poor resolution
a) Sample volume too large. Concentrate samples.
b) Excess micelle formation. Do not exceed 200 microgram SDS/30 microliter sample.
Problem 2: Run taking unusually long time
a) Buffers too concentrated. Check buffer protocol; dilute buffer if necessary.
b) Current too low. Increase voltage by 25-50%.
Problem 3: Run too fast, poor resolution
a) Buffers too dilute. Check buffer protocol; concentrate buffer if necessary.
b) Current too high. Decrease voltage by 25-50%.
Problem 4: More bands than expected observed for a purified protein
a) Proteolysis. Minimize the time between sample preparation and electrophoresis.
Problem 5: Fewer bands than expected with a heavy band at the dye front
a) Gel percentage is too low for the molecular weight range of the protein sample. Use a higher percentage acrylamide gel (increase % T in resolving gel).
Problem 6: Doublets observed where a single protein band is expected on SDS-PAGE
a) A portion of the protein sample may have re-oxidised during the run, or may not have been fully reduced prior to run. Prepare fresh sample solution using fresh ß-mercaptoethanol or dithiothrietol (DTT). Increase ß-mercaptoethanol or DTT concentration in the sample buffer.
Problem 7: Artifact band observed at approx. 67kDa in reduced samples, especially with silver staining
a) Excess reducing agent (ß-mercaptoethanol). The addition of iodoacetamide to the equilibration buffer just before applying the sample to the gel has been shown to eliminate these artifact bands.
b) Skin protein contaminants. Use new electrophoretic solutions and wear gloves when handling and loading the gel. More common when highly sensitive stains are used.
Problem 8: Skewed or distorted bands
a) Poor polymerization around sample wells. Increase ammonium persulfate and TEMED concentrations by 25%.
b) High salt concentration in sample. Remove by dialysis, Sephadex G-25 or any other desalting column or by Amicon concentrators.
c) Excessive pressure applied to the gel plates when the gel is placed into the clamp assembly. Do not overtighten the screws on the clamp assembly.
d) Uneven gel interface. Use a spirit level to make sure the gel apparatus is even. Overlay separating gel with water carefully.
e) Uneven heating of the gel. Either use a cooled apparatus or reduce the current at which electrophoresis is performed.
f) Insoluble material in the gel or inconsistent pore size throughout gel. Filter gel reagents before use and ensure that the gel mixture is well mixed and degassed before pouring the gel.
Problem 9: Lateral band spreading
a) Diffusion of sample out of the wells before the power was turned on. Minimize the time between sample application and power start-up.
b) Diffusion during migration through the stacking gel. Increase voltage by 25% during stacking gel or increase %T of stacking gel by 1%.
Problem 10: Vertical streaking of protein
a) Sample precipitation. Centrifuge all samples before loading wells. If problem still persists decrease %T of separating gel.
b) Sample overload. Dilute sample or reduce voltage by about 25% to minimize streaking.
Problem 11: Protein band curves upward at both sides of the gel. "Smile effect"
a) Center of the gel running hotter than either ends. Decrease power setting. Check buffer protocol to ensure it is properly formulated.
Problem 12: Same protein observed in several neighboring lanes
a) Samples from one well has contaminated adjacent wells. Use a Hamilton syringe to load wells and reduce the sample volume.
b) Do not delay while loading wells. A full well left next to an empty well would eventually contaminate the empty well over time.
Problem 13: Diffuse tracking dye
a) Decomposition of sample solution and/or buffer stock solution. Prepare fresh reagents.
Problem 14: Diffuse protein bands
a) Diffusion due to slow migration. Increase voltage by 25-50%. Check buffer to ensure it was properly prepared.
b) SDS or sample buffer too old. Prepare fresh solutions.
c) Protein sample not equilibrated. Equilibrate sample to running conditions.
d) Poor quality acrylamide or by resulting in incomplete catalysis. Use electrophoresis grade reagents.
e) Problems in sample preparation. Check to make sure sample is heated to at least 90°C for 2 min before loading.
Problem 15: Inconsistent relative mobilities
a) Incomplete catalysis. Excessive TEMED or ammonium persulfate. TEMED and ammonium persulfate should be 0.05%.
b) The constituents of the gel may vary in quality from batch to batch or with age. Use one batch of a chemical for as long as possible. Replace aged stock solutions and reagents.
c) The protein amounts loaded differ greatly. Do not overload the gel. Keep the loadings roughly similar in size.
Problem 16: Aggregation of proteins
a) Some samples aggregate on boiling. Treat sample at lower temperature (60°C).
b) Formation of disulfide bonds between protein in a complex mixture because of insufficient reducing agent. Prepare new sample buffer.
Problem 17: Band Streaking
a) High salt concentration. Precipitate and resuspend in lower salt buffer.
b) Sample too concentrated or not enough SDS. Dilute the sample with more SDS solution.
Problem 18: Heavily Stained band at gel origin
a) Gel concentration too high. Use lower gel concentration.
b) Aggregation of protein sample prior to electrophoresis. Refer to no. 16.
Problem 19: Aberrant Molecular Weight determination High molecular weight
a) Proteins with >10% carbohydrate bind less SDS. Check for carbohydrate.
b) Hydrophilic proteins bind less SDS.
c) Potassium or divalent cations present in sample precipitate SDS. Precipitate sample and resuspend in different buffer.
Problem 20: Aberrant Molecular Weight determination Lower Molecular Weight
a) Hydrophobic proteins bind more SDS.
b) Incomplete disulfide bond dissociation because of insufficient reducing agent. Prepare new sample buffer and samples.
Problem 21: Gelling time too long
a) Too little ammonium persulfate or TEMED. Increase both by 50%.
b) Temperature too low. Cast at room temperature.
c) Old ammonium persulfate and TEMED. Use fresh ammonium persulfate and new TEMED.
d) Poor quality acrylamide or bis. Use electrophoresis grade acrylamide and bis.
e) High concentration of thiol reagents. High concentrations of thiol reagent inhibit polymerization. Use less thiol reagent.
f) Omission of a reagent from the gel mixture. Have a list of all the reagents required and thick off reagent when utilized.
g) Incorrect concentrations of prepared reagents. Check protocol. Make up new reagents.
h) Degassing the acrylamide solution leads to a more rapid polymerization. However I have found that this step is cumbersome and unnecessary for most applications.
Problem 22: Gel too soft
a) Poor quality acrylamide or bis. Use electrophoresis grade acrylamide and bis.
b) Too little crosslinker. Make sure proper %C.
Problem 23: Gel does not polymerize
a) Temperature too low. Cast at room temperature.
b) Too little ammonium persulfate or TEMED. Increase both by 50%.
c) Poor quality acrylamide or bis. Use electrophoresis grade acrylamide and bis.
d) Ammonium persulfate or TEMED are old. Use fresh ammonium persulfate and new TEMED.
Problem 24: Swirls in gel
a) Excessive catalysis. Gel polymerizes in less than 15 minutes. Reduce ammonium persulfate and TEMED by 25% each.
b) Gel inhibition. Polymerization time >1 hour. Increase ammonium persulfate and TEMED by 50%.
Problem 25: Gel brittle
a) Too much crosslinker. Check protocol for %C. Recheck solution and weights.
Problem 26: Gel turns white
a) Bis concentration too high. Recheck solution or weights used.
Problem 27: Upper buffer chamber leaks
a) Upper buffer chamber over filled.
b) Improper assembly. Check assembly.
Problem 28: Leaking during gel casting
a) Chipped glass plates. Check glass plates for flaws. If minor flaws at bottom of glass plate parafilm could be used to properly seal the glass plates.
b) Improper alignment of gel plates. Check to ensure that the spacers and plate bottoms are flush.
Problem 29: Gel cracking during polymerization
a) Excess heat generation. Use cooled reagents.
Problem 30: Samples do not sink to bottom of well
a) Insufficient glycerol in the sample buffer. Recheck protocol.
b) Combs removed before stacking gel properly polymerized. Let stacking gel polymerized for 30 minutes before removing combs.
Problem 31: Sample preparation yellow in colour
a) Solution acidic: add NaOH until the solution turn blue.
b) Too little bromophenol blue in sample buffer.
Problem 32: Detachment of slab gels from glass plates during gel electrophoresis.
a) Inadequately cleaned glass plates
Problem 33: Base of sample well appears to be dragged downwards in the direction of electrophoresis.
a) Could be due to trapping of high molecular mass, high charged species at the gel surface. Very common when high concentrations of nucleic acid is present in the sample.
b) Check sample for nucleic acid and remove if present in significant quantities in the sample.
Problem 34: Poor Sample Wells
a) Distorted or broken wells are formed when the comb is not removed carefully. Comb should be removed only in a vertical manner.
b) When stacking gel resists the removal of the comb use a gel of lower % T.
c) When the wells contain a loose webbing of polyacrylamide it is likely that the comb fits loosely or the gelling rate is too fast. Replace the comb with a tighter fitting one and check the amount of TEMED and ammonium persulfate being added.
Problem 35: Gel Cracking during electrophoresis
a) The running conditions are too warm. This is especially common with high percentage gels.
Problem 36: Detachment of the gel from the glass plates
a) Probably due to unclean plates. After being rinsed with distilled water, they must drain cleanly without water spots.
Problem 37: Bands on part of the slab do not move down the gel
a) This is usually due to air bubbles between the plates underneath the affected lanes. Make sure no bubbles are present in the gel when pouring.
Problem 38: Formation of a sticky top on the gel
a) Penetration of the gel by butan-2-ol. Overlay the gel with butan-2-ol without mixing them. Do not leave butanol-2-ol to stand overnight on a polymerized gel or use water instead of butan-2-ol.
Problem 39: Protein bands are not sufficiently resolved
a) Insufficient electrophoresis. Prolong the run.
b) The separating gel's pore size is incorrect for the proteins that need to be separated. Alter the %T and/or %C of the separating gel appropriately.
Problem 40: Protein bands are not of uniform thickness
a) The sample was loaded unevenly. Check that the sample well bottoms are straight and horizontal.
Problem 41: Non-specific Coomassie blue staining
a) Decomposition of undissolved dye. Filter dye solution.
Problem 42: Protein bands not seen properly
a) Coomassie stain not sensitive enough. Gel can be rinsed and subsequently silver stained.
b) Not enough protein loaded onto the gel. For Coomassie blue stained gels each protein band needs at least 0.5 microgram of protein to be sufficiently stained.
c) Volume of Coomassie Blue too little. Increase the volume of staining solution to dilute out the SDS present in the gel.
d) Use a more concentrated staining solution and longer staining time.
e) Check the concentration of methanol (which strips SDS from the protein) used in the staining solution. Increase the methanol concentration if necessary.
Problem 43: Uneven staining of gels
a) Incomplete penetration of the dye. Leave gel in stain for a longer time.
b) Not enough dye.
c) Agitation was insufficient. Agitate when staining.
d) High concentration of SDS may interfere with coomassie blue staining.
Problem 44: Metallic sheen on gels after staining with Coomassie Blue
a) Solvent was allowed to evaporate causing the dye to dry on the gel.
Problem 45: A thin layer of Coomassie Blue on gel surface after destaining
a) Can easily be removed by a quick rinse in 50% methanol or by gently swabbing the gel surface with destain-soaked tissue paper.
Problem 46: Blotches near gel borders and over gel
a) Could be due to gel handling without gloves
Problem 47: Continuous stained region from the gel origin to near the buffer front.
a) Contamination of sample buffer. Make fresh sample buffer.
Problem 48: High silver staining background
a) Acrylic acid contamination in the acrylamide and/or bis-acrylamide. The highest quality reagents should be used.
b) Backgroung staining that is associated with silver detection of proteins in polyacrylamide gels has been shown to be due mostly to the amide groups in the crosslinker bisacrylamide (4). If the background staining is a serious problem use diacrylylpiperazine as the crosslinker. This crosslinker provides improved electrophoretic separation of proteins together with reduced background.
Problem 49: Stained bands become decolourized
a) Over-destained gel. Restain the gel and reduce the destaining time.
Problem 50: Cracking of gels during drying under vacuum
a) Vacuum released before gel is properly dry.
b) Slab gels > 1.5mm are being used.
c) Gel was allowed to swell before drying.
4) What are the common problems encountered in cell culturing? What are the causes of the problems? How to solve them?
Problem 1: No Viable Cells After Thawing Stock
Reason Solution Cells were stored incorrectly
- Obtain new stock and store in liquid nitrogen. Keep the cells in liquid nitrogen until thawing
Home made freezer stock is not viable
- Freeze cells at a density recommended by the supplier
- Use low-passage cells to make your own freezer stocks
- Follow procedures for freezing cells exactly as recommended by the supplier. Note the freezing procedure recommended by this handbook is a general procedure provided as a guideline only.
- Obtain new stock
Cells were thawed incorrectly
- Follow procedures for thawing cells exactly as recommended by the supplier. Note the thawing procedure recommended by this handbook is a general procedure provided as a guideline only.
- Make sure that you thaw the frozen cells quickly, but dilute them slowly using pre-warmed growth medium before plating.
Thawing medium is not correct
- Use the medium recommended but the supplier. Make sure the medium is pre-warmed
Cells are too dilute
- Plate thawed cells at high density as recommended by the supplier to optimize recovery.
Cells not handled gently
- Freezing and thawing procedures are stressful to most cells. Do notvortex, bang the flasks to dislodge the cells (except when culturing insect cells), or centrifuge the cells at high speeds.
Glycerol used in the freezing medium was stored in light (if applicable)
- If stored in light, glycerol is gets converted to acrolein, which toxic to cells. Obtain new stock.
Problem 2: Cells Grow Slowly
Reason Solution Growth medium is not correct
- Use pre-warmed growth medium as recommended by the supplier.
Serum in the growth medium is of poor quality
- Use serum from a different lot.
Cells have been passaged too many times
- Use healthy, low passage-number cells.
Cells were allowed to grow beyond confluency
- Passage mammalian cells when they are in the log-phase before they reach confluence.
Culture is contaminated with mycoplasma
- Discard cells, media, and reagents. Obtain new stock of cells, and use them with fresh media and reagents.
Problem 3: Rapid pH shift in medium
Possible Cause Suggested Solution Incorrect carbon dioxide (CO2) tension
- Increase or decrease percentage of CO2 in the incubator based on concentration of sodium bicarbonate in medium.
- For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, use CO2 amounts of 5% to 10%, respectively. Switch to CO2-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 to 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. (See Decontaminating Cultures with Antibiotics and Antimycotics.)
Problem 4: Precipitate in medium, no change in pH
Possible Cause Suggested Solution Residual phosphate left over from detergent washing, which may precipitate powdered medium components
- Rinse glassware in deionized, distilled water several times, then sterilize.
- Warm medium to 37°C and swirl to dissolve. If precipitate remains, discard medium.
Problem 5: Precipitate in medium, change in pH
Possible Cause Suggested Solution Bacterial or fungal contamination
- Discard medium.
- Try to decontaminate culture. (See Decontaminating Cultures with Antibiotics and Antimycotics.)
Problem 6: Cells not adhering to culture vessel
Possible Cause Suggested Solution Overly trypsinized cells
- Trypsinize for a shorter time, or use less trypsin. (See Dissociation of Cells from Culture Vessels.)
- Segregate culture and test for mycoplasma infection. Clean hood and incubator. If culture is contaminated, discard.
No attachment factors in medium
- For serum-free formulations, be sure they contain attachment factors.
Problem 7: Decreased growth of culture
Possible Cause Suggested Solution Change in medium or serum
- Compare media formulations for differences in glucose, amino acids, and other components.
- Compare the old lot of serum with the new lot in a growth experiment.
- Increase initial cell inoculum.
- Adapt cells sequentially to new medium.
5) What are the common DNA sequencing problems encountered? What are the causes of the problems? How to solve them?
Problem 1: Short DNA sequencing read lengths
- Too much template DNA
- Excessive dilution of the BigDye reagent
- Too little DNA
- Too much primer
- "Dirty" template DNA has been used.
- Unsequencable region reached (eg. homopolymer G)
Check the concentration of the template DNA by gel electrophoresis. Do not rely on spectrophotometer readings alone as spec reading are often inaccurate, particular with plasmid templates.
Reduce the reaction scale rather than using a higher bigdye dilution. It is better to reduce the reaction volume rather than use very high dilution factors of the BigDye chemistry. For example, a 1:16 dilution of the BigDye (0.5 µl total) can be achieve with modest dilution (1:4) by performing the reaction in a total volume of 5µl.
Check to see if an unsequencable region has been reached. If an unsequencable or “hard stop” region is the cause then it may be possible to sequence though it by PCR amplifying the region using 7-deaza-deoxy guanosine triphosphate (7-deaza-dGTP), then sequencing the PCR product directly. The 7-deaza-dGTP analog disrupts the hoogsteen base pairing between successive guanosine bases and allow the sequencing DNA polymerase through the high G+C region.
Check that the oligonucleotide primer concentration is correct. Do not rely on old primer stocks, especially those made up by your lab colleagues!
Check that the template is clean. Consider sequencing a PCR amplified insert or switching to using a commercial plasmid miniprep kit if you are finding that many of your sequencing reactions are failing.
Problem 2: Mixed template sequencing traces
- Two or more templates were present in the reaction. This is the most common cause of mixed template.
- A "double pick" of two colonies. This can when the colonies are too close together on the colony plate.
- Two primers were present in the sequencing reactions. This can occur when using premixed PCR regents for sequencing where the primer stock is actually a mix of universal forward and reverse oligonucleotide primers.
- The PCR fragment was not purified of leftover primes before sequencing.
- Two priming sites are present in DNA template. This can occur when a PCR product with universal priming site tails is cloned into a plasmid.
- Poor quality PCR template containing multiple DNA fragments was used.
- Too low a primer annealing temperature was used in the sequencing reaction.
- Different sequencing reactions were accidentally mixed at the clean up stage. This can also sometime occur if the same tip is used without rinsing.
Makes sure only one DNA template is present. Prepare a new plasmid prep making sure that only one colony is selected. If sequencing a PCR product check that only one PCR product is present by running an agarose gel. Remember that even a relatively low amount of a small PCR product can cause mixed template problems. Check the template for possible multiple priming sites. If two site are present use a different primer. This can often occur when a fragment contain the priming is sub-cloned into a vector that also contains the priming site. Be very careful of this problem when using the M13 universal primers.
Insure that you use a PCR clean-up protocol that remove leftover PCR primers. Even low levels of the PCR primers can cause mixed signal problems, especially if they have a high annealing temperature.
Check the predicted melting temperature of the sequencing primer. If it is more than 5˚C above the annealing temperature used in the sequencing reaction then raise the annealing temperature. Because of the inclusion dITP in the BigDye sequencing mix the annealing/extension temperature can't be raised above 60˚C. If your primer is still misanneals at 60˚C then synthesize a new primer (this is easily done by removing bases from the 5' end until the Tm is below 60˚C).
Problem 3: Failed DNA sequencing reactions
- Poor quality DNA. Very common when sequencing plasmid miniprep templates.
- Loss of the reaction during clean up. This can be a particular problem when using ethanol precipitation clean up protocols.
- Too much template DNA. Excess template can kill the sequencing reaction.
- Wrong primer used. More common than you might think!
- Bad water. The water used contains a sequencing inhibitor.
- Degraded or failed synthesis primer. Oligonucleotide synthesis is chemically complex and primer synthesis failures is fairly common.
- Dead sequencing chemistry. Can occur if the BigDye chemistry is stored under the wrong conditions or is freeze-thawed too many times. Either the TaqDNA polymerase or dye labeled nucleotides can have degraded.
- Blocked capillary. Every trace using a particular capillary fails. Can be identified by tracking trace quality on a trace by trace basis.
Poor quality DNA. The best way of avoiding this problem is to not sequence plasmid DNA and sequence a PCR amplified fragment of the plasmid insert. If this is not possible then it is recommended that a plasmid miniprep kit is used. One tip is to perform a final ethanol precipitation on the kit purified plasmid DNA. This often solves problems with the quality of the template.
Loss of sequencing reaction during clean up. This can be avoided by not using an ethanol precipitation protocol to clean up the sequencing reaction. There are a number of kits that work very well, unfortunately they can be very expensive. One tip for avoiding loss of the reaction DNA pellet when using the ethanol protocol is to add 1µl of a 20 mg/ml solution of glycogen (Sigma G-1508) to the sequencing reaction before adding the ethanol. This helps make the pellet visible and the glycogen does not seem to interfere with the injection of the sequencing fragments onto the sequencers capillaries.
Too much template DNA was used. This can be avoid by checking the concentration of the template on an agarose gel before sequencing. This will also allow you to see the purity of the template DNA and if there is a significant amount of contaminating genomic DNA or RNA present. Do not rely on a spectrophotometer reading to calculate the template concentration.
Wrong primer. This is simple to solve, but can be difficult to detect. Check the sequence of the primer and template to make sure that the primer binding site is present. This can be a particular problem with some "universal" forward and reverse primer sequences which do not work with some common plasmids. Do not trust other people's working stock solutions (especially those of your unreliable your lab colleagues!) and make your own. It might take 5 minutes longer, but it will save you a lot of future headaches.
Bad water. Inhibitors can end up in lab water stocks that can kill DNA sequencing reactions. If you think this may be a problem then throw out the water and use a fresh stock - remember water is cheap.
Degraded primer. Don't use old diluted primer stocks. Store the primers in 10mM Tris/ 0.1 mM EDTA (pH 8.5) rather than water. Don't use other peoples stocks. If you have any doubt about how the primer quality through it out and make up a fresh working solution from the primer stock.
Failed oligonucleotide synthesis. If you suspect that the primer is poor quality either have it presbyters or check in a polymerase chain reaction (PCR). Alternatively if you have a control template that you know should work with the primer then this can be a good way of identifying primer problems.
Dead sequencing chemistry. This is a relatively rare problem, however, if a batch of BigDye chemistry has not been used for some time, or there is any doubt about how it has been stored, then it is advisable to perform a control sequencing reaction before undertaking a large number of experimental reactions. Many problems with dead chemistry can be prevented by storing the BigDye chemistry in small aliquots and avoiding repeated freeze/thaw cycles.
Blocked capillary. Can be identified by tracking trace quality on a trace by trace basis. While this can be done manually we recommend using our QualTrace software. If you suspect that a sequencer capillary is blocked then inform the operator.
Problem 4: Delayed signal start sequencing traces
Capillary overload. Unlike a lot of DNA sequencing problems delayed peak signal has only one cause - capillary overload. The two most common causes of capillary overload are too much template DNA or dirty template DNA contaminated with proteins an/or salt.
Use less template DNA. Modern ABI capillary sequencers require far less template DNA than the old slab based sequencers, however, many researchers are still using protocols developed for the slab based system. Try the suggestions made in the DNA sequencing protocol tips page.
Use cleaner DNA. Try changing you template preparation protocol. A good cheap template preparation protocol that works well with sequencing is the ExoI/SAP clean up.
6) Can I reuse DNA samples which have been measured using spectrophotometer for downstream processing?
According to research, reusing DNA samples for downstream applications is possible, some models of spectrophotometer are capable of maintaining the quality of the DNA sample, for example, Eppendorf BioPhotometer plus. DNA samples were measured multiple times in three different concentrations using the BioPhotometer plus and subsequently employed directly in real-time PCR and standard PCR experiments. These experiments did not detect any loss of quality of the DNA due to UV light as compared to unmeasured controls. Based on these results, we can prove that reusing DNA sample which have been measured using spectrophotometer for downstream processing is possible.
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7) Why do I get low efficiency of DNA amplification when I have used filter tips for preparation of PCR experiments?
This is because the use of various filter tips for preparation of PCR experiments can lead to significant decreases in the efficiency of DNA amplification, when the filter material comes into contact with the PCR sample. Due to filter contact, general adhesive effects can cause specific sample agents to bind to the filter material and causes them to be no longer available for the DNA amplification process. Moreover, many filter tips contain self-sealing additives, which may dissolve when they come into contact with an aqueous phase, thus contaminating the sample. These factors are another source of potential losses in efficiency during a PCR experiment.
Non-self-sealing filter tips will be able to solve this problem, such as the Eppendorf epDualfilter T.I.P.S., where contamination due to self-sealing additives is impossible.
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Beside checking your transfection reagent, you must first check what type of plasmid extraction kit you are using. It MUST be transfection-grade plasmid DNA kit such as 5Prime PerfectPrep EndoFree Plasmid kit. It utilizes an endotoxin removal process to generate high quality plasmid DNA for use in advanced downstream applications.
Efficient endotoxin removal (<0.1 endotoxin units per ug plasmid DNA) improves mammalian cell viability. The kit includes PerfectPrep EndoFree Filter CS. The resulting plasmid DNS is highly suited for use in a broad variety of demanding applications, including transfection of sensitive and primary cell lines, in-vitro transcription and translation, and all enzymatic modifications.
High concentration of ammonia is use to break oligos/primer from the support linkage after oligo synthesis. Therefore, we need to do further purification to get rid of the salt.
PCR grade primer is purified by ethanol precipitation, where the purity is very poor (only 60-70%). It is only suitable for normal PCR.
HAP purified primer is purified using affinity column where the purity is 98%. It is suitable for PCR and sequencing. PAGE and HPLC purified primer or probe can achieve up to 99% purity. They are suitable for all high-end PCR work, including, SNP and real-time PCR.
When working with RNA it is essential to avoid contamination with RNase.
- Always wear and change disposable gloves. Do not simply touch anything after wearing the gloves such as door handle, cabinet handle, etc. to avoid RNase contamination.
- Use sterile, disposable plasticware and micropipettes reserved only for RNA work to prevent cross contamination of RNases from shared equipment. NEVER share your pipette with DNA work as DNA extraction often uses RNase.
- Glass items can be baked at 150℃ for 4 hours. Autoclaving will not get rid of RNase.
- Plastic items should be soaked in 0.5 M NaOH for 10 min, rinsed thoroughly with water, and then autoclaved.
- Treat water with 0.01 % (v/v) DEPC overnight in bottle and autoclave the next day.
- Do not share reagents and bench top for RNA work.
- Good quality RNA will have an OD 260/280 ratio (purity) of 1.8 to 2.
- Avoid repetitive freezing and thawing.
T4 DNA ligase is having two types of unit definition; Weiss Unit and Cohesive end ligation unit, depending on the supplier. The unit stated in most of the reference books is in Weiss unit, where one Weiss unit is equivalent to 67 Cohesive End ligation units. Therefore, it is a must check the product description before adding the unit concentration.