I received my primer order, but the yield is lower than the scale that I ordered. Why is this?
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
Im getting low yield of my oligo upon reconstitution. What happened?
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
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?
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
Im missing a nucleotide in the middle of my sequence. How could this happen?
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
My primer has an extra inserted base. How could this happen?
I just received my primers and they look yellow. Can I still use them?
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
There is a green color in my lyophilized oligo. Can I still use it?
There is a ball-shaped pellet at the bottom of my oligo tube. What is this and can I still use my oligo?
I dont see a pellet in my oligo tube order. Should I ask for a replacement?
The primers I am using worked for PCR initially, but over time, have stopped working. What happened?
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
My oligonucleotide does not appear to be the right length when I checked by gel electrophoresis. Why is this?
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. dCs migrate fastest, followed by dAs, dTs, and then dGs. Oligos containing Ns tend to run as a blurry band and generally have a problem with secondary structure.
Answer Id: 7303
What are the main steps in PCR?
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
What does hot start PCR mean?
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
Why is it difficult to amplify a GC-rich template?
How can I facilitate the amplification of templates with hairpin-loop structures or high GC-content?