How do you reconstitute primers from IDT?
To obtain a 100 µM solution, multiply # nmol x 10. That will equal the # µL to use for resuspension. For example: 20 nmol X 10 = 200 µL.
What concentration are IDT primers?
What are the concentrations of Custom NGS Adapters and Indexing Primers?
| Adapter or primer | Final concentration (µM) |
|---|---|
| TruSeq™–Compatible Stubby Adapters (use with TruSeq™–Compatible Primers) | 15 |
| TruSeq™–Compatible Primers (use wtih TruSeq™–Compatible Stubby Adapters) | 10 |
| Nextera™–Compatible Indexing Primers | 10 |
Why might it be advantageous to include degenerate sites in the primers?
A degenerate primer is mixture of primers that has substitution of different bases sequence (they are similar not same). They are usefull if need to amplify a gene from similar organism. So it possible amplify different sequence which represent different protein sequence.
What is a degenerate sequence?
A PCR primer sequence is called degenerate if some of its positions have several possible bases. The degeneracy of the primer is the number of unique sequence combinations it contains. We study the problem of designing a pair of primers with prescribed degeneracy that match a maximum number of given input sequences.
Is it OK to vortex primers?
Don’t vortex too much. But you can vortex gently for 5-10 for proper mixing. It would not break primers.
Can I resuspend primers in water?
Note: For long-term storage, avoid resuspending oligos in DEPC-treated water or water from deionizing systems. These are often acidic (pH as low as 5) and may cause DNA degradation over time.
What happens when you increase degeneracy in primers?
However, high degeneracy increases the chance that primers will also bind to non-target regions13. While sequences from non-target regions can be filtered out bioinformatically or by size selection of PCR products (assuming different amplicon lengths), such filtration can reduce the yield of target fragments.
What are degenerate primers and why would you use them?
Degenerate primer design. Degenerate primers are useful for pulling out one part of a gene sequence when you only know the gene sequence in related organisms. The more distant those related organsims, the more difficult it can be to design primers.
What are the challenges of designing primers?
Poor design choices, erroneous or truncated sequences, and ineffective purification can lead to unusable results. We have applied thermodynamic and bioinformatic knowledge towards a suite of easy-to-use, online tools to help you design primers.
What are mixed bases in IDT?
Mixed bases IDT offers oligonucleotides containing randomized or “mixed” bases. The final order contains a population of oligos with a different base at the randomized position. Mixed bases are ideal for creating pools of oligos that include matches to a variable or unknown template sequence.
What is the most important consideration when designing PCR primers?
Custom primers The accuracy of design and synthesis of a primer pair is the most important consideration to generate good PCR performance data. Poor design choices, erroneous or truncated sequences, and ineffective purification can lead to unusable results.
How do I determine the T M value of PCR primers?
Therefore, to obtain the T m values for your specific experimental conditions, it is important that you use your own reaction parameters. IDT recommends that you aim for PCR primers between 18 and 30 bases; however, the most important considerations for primer design should be their T m value and specificity.