<b>Page under construction as of 2/22/2023</b> ---+ Designing Specific Primers on Benchling While there are tools available for automatically designing primers (such as the [[https://www.ncbi.nlm.nih.gov/tools/primer-blast/][NCBI Primer BLAST]]) often specific PCR applications, such as amplifying fragments for cloning, will necessitate specific primer placements and modifications. This page presents guidelines and tools for specifically designing primers from DNA sequences, using the Benchling platform as an example. In short, you will need to: </br><b>1)</b> determine where to place your primers within the template sequence to amplify your desired product </br><b>2)</b> adjust your primer sequences to ideal annealing temperatures </br><b>3)</b> modify the sequences with any necessary adapters and check for inhibitory secondary structures. ---++ 1) Determining Primer Placement ---++ 2) Calculating Annealing Temperatures Calculate the annealing temperature for your PCR reaction using the [[https://tmcalculator.neb.com/#!/main][NEB Tm Calculator]].</br> <b>a.</b> Select the polymerase for your PCR reaction from the dropdown menu.</br> <b>b.</b> Copy ~30 bp of the forward and reverse primer sequences into the Primer 1 and Primer 2 boxes.</br> <b>c.</b> Adjust the annealing temperature (top right box) by removing bases from the 3' end of each primer until:</br> * The annealing temperature is in your desired range. This is typically ~57-60°C. * The individual Tms of the two primers (shown in the boxes on the right) are less than 5° apart. For best results the individual Tms should be as close together as possible. * Each primer is 18-25 bp long. Shorter primers are preferred because they reduce the possibility of forming secondary structures that may inhibit your reaction. ---++ 3) Modifying Primer Sequences and Checking Secondary Structures https://www.idtdna.com/calc/analyzer ---++ Further Reading * [[https://www.benchling.com/primer-design-for-pcr#chapter4][Benchling primer design guidelines]]
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Topic revision: r1 - 2023-02-22 - IsaacGifford