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

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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: 7293

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7d91786c01b3931e8d94baf248608979_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: 7294

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bc047286b224b7bfa73d4cb02de1238d_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?

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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: 7295

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f953ad57910572bd6803da3faaa6e92b_FAQ

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

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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: 7296

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d7f14b4988c30cc40e5e7b7d157bc018_FAQ

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

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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: 7297

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142536b9b535b78e681c11b0195d962f_FAQ

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

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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: 7298

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cc360b61d7eb072c77a4beddebb3c95b_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: 7299

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29e11dc359bad383e1243f730bdbe032_FAQ

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

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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: 7300

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220787ad7829c9cbc7e9953cb1c36fb3_FAQ

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

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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: 7301

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9c779f56f336b3c812343434f57b6a0e_FAQ

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

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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: 7302

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d9437926cc8d785a7bdb8578fd85d8e3_FAQ

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

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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: 7303

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27debb435021eb68b3965290b5e24c49_FAQ

What are the main steps in PCR?

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Answer

The main steps are: denaturation, annealing, and extension. The template is typically heated to a high temperature (around 94-95 degrees C) allowing for the double-stranded DNA to denature into single strands. Next, the temperature is lowered to 50-65 degrees C, allowing primers to anneal to their complementary base-pair regions. The temperature is then increased to 72 degrees C, allowing for the polymerase to bind and synthesize a new strand of DNA.

Answer Id: 7269

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35d8f387d4934b6ee53ce5c9a1d8c1d7_FAQ

What does hot start PCR mean?

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Answer

Hot start is a way to prevent DNA amplification from occurring before you want it to. One way to do this is to set up the PCR reaction on ice, which prevents the DNA polymerase from being active. An easier method is a use a ‘hot-start’ enzyme, in which the DNA polymerase is provided in an inactive state until it undergoes a high-heat step.

Answer Id: 7270

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7f489f642a0ddb10272b5c31057f0663_FAQ

Why is it difficult to amplify a GC-rich template?

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Answer

A GC-rich template often has a higher melting temperature and may not denature completely under the normal reaction conditions.

Answer Id: 7272

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d2a1e34d86293cb12f959f89dddf263e_FAQ

How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content?

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Answer

You can try adding 5-10% DMSO, up to 10% glycerol, or 1-2% formamide or a combination of these to facilitate difficult templates. Note: the use of cosolvents will lower the optimal annealing temperatures of your primers.

Answer Id: 7273

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