What is a primer sequence?

A primer is a short, single-stranded DNA sequence used in the polymerase chain reaction (PCR) technique. In the PCR method, a pair of primers is used to hybridize with the sample DNA and define the region of the DNA that will be amplified.

Do primers go from 5 to 3?

Sequences are always written from 5′ to 3′. This includes the sequence of your template DNA (if known), the sequence of the vector DNA into which it is inserted, and the sequence of proposed primers. Don’t ever write a primer sequence reversed or you will only confuse yourself and others.

How many primers are needed for genotyping the T-DNA mutant?

three primers
Using three primers at a time (gene specific LP, RP, and a T-DNA border primer), clearly distinguishes homozygous, heterozygous, and wild type plants.

What are Salk lines?

T-DNA insertion lines provide important resource for genetic analysis based on mutagenesis in plant research. SALK lines are the most widely used T-DNA insertion lines for the model plant Arabidopsis. Accurate assessment of insertions is critical for understanding the value of the insertion lines.

How do you find the primer sequence?

You will start to get sequence ~20 bp downstream of your primer. If the PCR product is <800 bp then your sequence should run toward the opposing primer and will end around 5-10 bp from the end of your PCR product. From here you should be able to get an approximation of the primer binding site in your target gene.

How long should a sequencing primer be?

18 to 22 bases
Primer length should be in the range of 18 to 22 bases. The primer should have GC content of 50% to 55%. Primers should have a GC-lock on the 3′ end.

How do you find the forward primer?

The forward primer is easy and is the primer that resides on the bottom strand on the 3′ side. The reverse primer is more complicated and binds to the top strand on the 3′ side.

How far apart should primers be?

No primer-dimers greater than 6 bp between each primer and itself and also between the two primers. Forward and reverse primers should be about 500 bp apart. The 3′ end of the primer should be a G or a C. The genomic sequence that comes from the computer is just one strand; the complementary strand is not shown.

How do you design primers from a DNA sequence?

PCR Primer Design Tips

1. Aim for the GC content to be between 40 and 60% with the 3′ of a primer ending in G or C to promote binding.
2. A good length for PCR primers is generally around 18-30 bases.
3. Try to make the melting temperature (Tm) of the primers between 65°C and 75°C, and within 5°C of each other.

How many primers do you need for sequencing?

Two
Two PCR primers are needed in a PCR reaction (usually); only one sequencing primer is added to a sequencing reaction.