Selecting Primers 17-21 bp from cDNA Last Half

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In summary, when selecting primers for PCR, it is recommended to choose ones that are between 17-21 bp and located in the last half of the cDNA. This helps to reduce the likelihood of binding to random sequences and maintain proper temperatures during PCR. It is important to avoid short or AT-rich sequences, as this can lead to a loss of specificity for the DNA sequence.
  • #1
sobored
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It says that i have to pick primers between 17-21 bp which are found in the last half of the cDNA. How come the last part of the cDNA?


Thanks.
 
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  • #2
PCR protocols

I am no expert on primers. However I did find a professor at Penn State University who may be able to assist. Just follow my blue underlined hyperlink to his webpage on "Polymerase Chain Reaction Protocols. (His email address is on that page)

He refers to picking primers as you have inquired (see below)
"Using the Primer Selection Program...
...Keep in mind that we prefer to pick primers between 17-21 bp which are found in the last half of the cDNA and result in a product of 100-400 bp."
 
  • #3
you choose your primers differently depending on what you want to do
you usually use a oligomer around 20 nucleotides long because of it reduces the likelihood that the primer will bind to a random sequence and because you need to maintain certain temperatures for proper annealing and elongation during PCR. If your sequence is too short or too AT rich, then the primer will more likely be able to be denatured from your DNA during annealing and this causes a loss of specificity for your DNA sequence.
 

Related to Selecting Primers 17-21 bp from cDNA Last Half

What does "Selecting Primers 17-21 bp from cDNA Last Half" mean?

"Selecting Primers 17-21 bp from cDNA Last Half" refers to the process of choosing short segments of nucleotides (building blocks of DNA) that will be used to initiate the amplification of a specific section of cDNA (complementary DNA) in a laboratory setting.

Why is selecting the right primers important?

Selecting the right primers is crucial because they determine the specificity and efficiency of the amplification process. If the primers do not match the target cDNA sequence accurately, the desired amplification will not occur. Additionally, using primers that are too long or too short can result in non-specific amplification or low amplification efficiency, respectively.

What is the recommended length for primers in cDNA amplification?

The recommended length for primers in cDNA amplification is between 17-21 base pairs (bp). This length is considered optimal for ensuring specificity and efficiency of the amplification process, as well as minimizing the likelihood of primer-dimer formation.

How can one select the best primers for cDNA amplification?

To select the best primers for cDNA amplification, one should first identify the target cDNA sequence and then use specialized software or online tools to design primers that meet the recommended length and other criteria such as melting temperature, GC content, and absence of hairpin structures. It is also important to validate the primers through experimental testing.

What are some common challenges in selecting primers for cDNA amplification?

Some common challenges in selecting primers for cDNA amplification include finding primers that are specific to the target sequence, avoiding primer-dimer formation, and minimizing the presence of secondary structures. Additionally, the presence of repetitive sequences or genetic variations in the target cDNA can also pose challenges in primer selection.

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