I’m getting low yield of my desired fragment. What am I doing wrong and how can I increase my yield?

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Answer

Please see our suggestions below to increase yield:

-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR. [Lee (1995) BioTechniques 18:225].
-Not enough enzyme was used.
-Denaturation/extension temperature was too high and enzyme died prematurely.
-Too much DMSO (>10%).
-Incorrect annealing temperature: run a series of reactions using different annealing temperatures, starting 5 degrees below the calculated Tm.
-Too few cycles.
-Insufficient or too much Mg2+.
-Poorly designed primers: double check primer sequence against template sequence, primers should have similar melting temperatures, avoid complementary sequences at the 3’ end of primers.
-Carryover inhibitors (e.g., blood, serum).
-Denaturation time was too short. Genomic and viral DNA can require denaturation times of 10 minutes.
-Not a long enough extension time was used depending on the size of product being amplified.
-Use of super-irradiated (treated with >2500 mj/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR]. Alternatively, a combination of 1.0 M betaine with 6-8% DMSO or 5% DMSO with 1.2-1.8 M betaine can be used to amplify GC-rich templates [Baskaran (1996) Genome Res 6:633].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme, melanin, etc.). Add BSA to the PCR (~160-600 μg/mL), increase the amount of Taq, and/or increase the volume of the PCR to dilute out the inhibitor. The concentration of BSA to add may be dependent on the amount and type of inhibitor present. Additionally, fatty acid-free, alcohol-precipitated BSA, or Fraction V BSA all should be effective.

Answer Id: E7289

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fbc850f92dd2fb51e579ecde938942f0_FAQ

I’m getting no bands from my PCR product. What could cause this?

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Answer

Here are some reasons why your PCR experiment may be failing:

-NaCl at 50 mM will inhibit the enzyme.
-Too much KCl in the reaction. Do not exceed 50 mM.
-Incorrect annealing temperature was used.
-Incomplete denaturation (time and temperature must be long and high enough).
-Template had long runs of GC's [Woodford et al. (1995) Nucleic Acids Res 23:539 show that by eliminating all potassium from the amplification reactions, GC-rich regions in templates are sufficiently destabilized to allow PCR].
-10% DMSO partially inhibits Taq.
-Hemin (in blood samples) inhibits Taq.
-Use of super-irradiated (treated with >2500 mJ/cm2) mineral oil will either inhibit or decrease yield of PCR product [Dohner (1995) Biotechniques 18:964].
-Do not use a wooden toothpick to pick colonies or scoop out DNA from a gel prior to PCR. It has been reported that this technique can inhibit PCR [Lee (1995) BioTechniques 18:225].
-Other inhibitors of Taq DNA polymerase were present (e.g., indigo dyes, heme). Add BSA to the PCR, increase the amount of Taq, and/or increase the volume of the PCR to dilute out them inhibitor.

Answer Id: E7290

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d2e11a4bbd54bfddd44611e3a6a81518_FAQ

I’m getting an unexpected product when performing PCR. What could be the cause of this and what do you suggest I try?

Product FAQ

Answer

Please see the following possibilities and suggestions we have:

-Primer design: try longer primers to avoid binding at alternative sites, avoid 3 consecutive G or C nucleotides at the 3’ end.
-Annealing temperature: increase annealing temperature to increase specificity.
-Mg2+ concentration: try a lower concentration.
-DNA contamination: use aerosol tips and separate work area to avoid contamination, use UNG/UDG technique to prevent carryover.

Answer Id: E7291

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ae0d97e847df896ca90806eff2f0147b_FAQ

I’m seeing high molecular weight EtBr stainable material left in wells. Why is this happening?

Product FAQ

Answer

This artifact occurs when either too many cycles were performed or too much DNA is added to the reaction. Try heating to 65 degrees C and putting sample on ice before loading.

Answer Id: E7292

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e4f40a1c304c83f3110acc2eaa261b25_FAQ

I received my primer order, but the yield is lower than the scale that I ordered. Why is this?

Product FAQ

Answer

The scale that is ordered refers to the starting synthesis scale, or amount of starting material used to create your oligo. Based on purification and efficiency, you will receive less than the starting synthesis scale. However, we do have a minimum yield guarantee based on the starting synthesis scale which can be found here: https://www.lifetechnologies.com/us/en/home/products-and-services/product-types/primers-oligos-nucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/oligo-minimum-yield-guarantee.html.

Answer Id: E7293

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31f0370052f798324596bd2ee558cdb1_FAQ

I’m getting low yield of my oligo upon reconstitution. What happened?

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Answer

The oligo may not have been fully solubilized. After addition of TE buffer, make sure the oligo was vortexed for a full 30 seconds and/or pipette up and down more than 10 times. Primers may be present along the sides of the tubs, so when resuspending the oligo, the sides of the tubes should be “rinsed” too.

Answer Id: E7294

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76b3305be84782cc5852c019757c0382_FAQ

I ordered a primer with restriction enzyme sites flanking the 3’ and 5’ ends of my oligo with desalted purification. When trying to subclone the PCR product, I get very few colonies. I have tested all conditions, and it seems to be the oligo causing the problem. Can you explain why this happened?

Product FAQ

Answer

Better purification of the oligos is recommended to provide you with full-length oligo sequence. Adding restriction sites adds on 10 or more bases to the basic 20-25-mer, making primers longer than 30 bases with a relatively low percentage of full-length sequences after desalting. Additionally, failure sequences occur at the 5’ end of the sequence as oligos are generated from 3’ to 5’ end. Therefore, restriction sites introduced at the 5’ end of primers can be compromised, resulting in missing bases.

Answer Id: E7295

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59d47d44b31148aeb225418a3b31ca7a_FAQ

I’m missing a nucleotide in the middle of my sequence. How could this happen?

Product FAQ

Answer

There are two possibilities that could occur in any round of extension when creating your primer:

1.The added base is not detritylated correctly, missing one base addition but allowing possible extension in the next round.
2.The trityl group was removed, but not coupled or capped correctly before addition of the next base, allowing the chain to continue.

Answer Id: E7296

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ff7713e6113e25aa3655808616b408b8_FAQ

My primer has an extra inserted base. How could this happen?

Product FAQ

Answer

If detritylation occurs inappropriately and/or if the synthesizer has an error and delivers the wrong base, an extra inserted base can occur in your primer. Please contact techsupport@lifetech.com for assistance.

Answer Id: E7297

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d31ec4930d61346dd1c3bfd16325451b_FAQ

I just received my primers and they look yellow. Can I still use them?

Product FAQ

Answer

Most of the time the color should not affect PCR or any other experimental application since typically it is caused by the iodine used in the synthesis. There are some exceptions, however. Brown oligos can also be caused by the primer being overdried, and if this is the case, the primer may not work.

Answer Id: E7298

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da3f0afb47dc65696e92d77bab276db2_FAQ

There is a green color in my lyophilized oligo. Can I still use it?

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Answer

If an oligo appears green in color, this is most likely due to ink falling into the tube. The oligo should still be fully functional. The color can be removed by doing an ethanol precipitation.

Answer Id: E7299

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54dc07392bd721ba4eaa8379460ee44e_FAQ

There is a ball-shaped pellet at the bottom of my oligo tube. What is this and can I still use my oligo?

Product FAQ

Answer

If the oligo was overheated, it will appear as a “ball”-shaped pellet attached to the bottom of the tube. This should not affect the quality of the oligo, and the oligo should be readily soluble in water.

Answer Id: E7300

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46aaecde18c51d0a6f358ac12f960123_FAQ

I don’t see a pellet in my oligo tube order. Should I ask for a replacement?

Product FAQ

Answer

The drying method dries the primer in a thin layer along the sidewalls of the tube instead of the bottom, therefore a pellet is not always visible and should still be ready to use.

Answer Id: E7301

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ef36da5a1aca15ca08a8adfb63452558_FAQ

The primers I am using worked for PCR initially, but over time, have stopped working. What happened?

Product FAQ

Answer

Primers should be aliquoted for single use before PCR set-up. Heat just the aliquoted primers to 94 degrees for 1 min. Quick chill the primer on ice before adding to the PCR reaction. Some primers may anneal to themselves or curl up on themselves.

Answer Id: E7302

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03de3389616245442315d9da95048050_FAQ

My oligonucleotide does not appear to be the right length when I checked by gel electrophoresis. Why is this?

Product FAQ

Answer

Oligos should be run on a polyacrylamide gel containing 7 M urea and loaded with a 50% formamide solution to avoid compressions and secondary structures. Oligos of the same length and different compositions can electrophorese differently. dC’s migrate fastest, followed by dA’s, dT’s, and then dG’s. Oligos containing N’s tend to run as a blurry band and generally have a problem with secondary structure.

Answer Id: E7303

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26b0014ea075f0ddca60a6029acb593b_FAQ