Primer Design Tool for the 1st PCR and Instruction of how to use this tool

Purpose of developing the primer design tool for the 1st PCR

The Primer Design Tool for the 1st PCR is available to download at the bottom of the page.

The 2-step PCR is the first step in the protocol of CFPS700 kit to generate the DNA template to be used in the following transcription and translation. The 1st PCR aims to connect the target ORF sequence with the 2nd forward/reverse primers (please see the diagram below). Therefore, the 1st PCR forward/reverse primers need to be target gene specific that overlap with the N-terminal and C-terminal sequences of the ORF of the target gene. There are two sets of forward and reverse primer sets in the 2nd PCR. The two reverse 2nd PCR primers and one forward 2nd PCR primers are universal primers. The other forward 2nd PCR primer contains a tag sequence, such as His-, FLAG-, or MYC-tag, therefore can differ from one another depending on your tag choice.

In the CFPS700 protocol, overlapping length between 1st gene specific primers and ORF is set at 18 bp as the default. But the overlapping length can be extended to 30 bps, giving multiple options for primer. For some options, depending on the GC content of N- or C-terminal sequences of ORF, Tm value difference between one 1st gene specific forward primer candidate and 1st gene specific reverse primer candidate can be higher than optimal. When this happens, the selection of this primer set would result in less efficient or even failed 1st PCR, subsequently 2nd PCR, and ultimately failed protein translation. Therefore, carefully selecting the optimal 1st PCR primer set with the minimum Tm value difference is very important to the success of both the 1st and 2nd PCRs and ultimately protein translation using CFPS700 kit. 

“Primer Design Tool for the 1st PCR” helps you select the best primer set for the 1st PCR. This tool calculates and shows the Tm value for each primer (forward or reverse) at different overlapping lengths, ranging between 18 bp and 30 bp. So you can select the best pair of 1st-gene-specific forward and reverse primers that have the minimum Tm value difference. Please note that the primers’ overlapping lengths with the N- or C-terminal sequences of the ORF do not need to be the same

primer design tool for the 1st PCR

Step by Step Instruction

Please follow the steps below to calculate and generate appropriate first forward and reverse primes.

  1. Specify which terminus to put the tag at “Tag” field.

    Select “C-term” or “N-term” from drop down list, which terminus of the target protein you want to put the tag to.

    When you will select “C-term”, 1st forward primer is “1st-gene-specific-CF” that is made of part of the OMEGA sequence and the gene specific forward primer sequence you will choose below; whereas the 1st reverse primer is “1st-gene-specific-CR”, composed of the linker sequence and the gene specific reverse sequence you will choose below (please refer to Table 4 in the CFPS700 protocol). When you select “N-term”, 1st forward primer is “1st-gene-specific-NF” and 1st reverse primer is “1st-gene-specific-NR” in the protocol (please refer to Table 2 in the CFPS700 protocol). The sequences shown in the two background boxes are for showing purpose.
    Note: Sequence of reverse primers is a complement sequence

  2. Copy and Paste ORF sequence at “ORF” field.
    Copy and paste the ORF sequence which starts from ATG and does not include stop codon at the end. Sequence string can include space, CR and/or LF.

    Then you will see the length in mer at the bottom.

  3. Modify Na+ and Primer concentration at “PCR Condition” field.
    These values are used for calculating Tm values. By default, concentration of Na+ is 50 mM and concentration of Primer is 500 nM. Generally, you do not need to modify these values. But you can modify these values according to your experiments or protocol of PCR enzymes.

  4. Specify which calculation method to be used at “Tm calc method” field

    You can specify Tm calculation method indicated as “GC” or “Nearest Neighbor” from drop down list.

    Please follow the method which your PCR enzyme uses in its manual. If it does not, Specify “GC” as default.

  5. Check the Tm value and select of the 1st-gene-specific-CF and1st-gene-specific-CR primers, or 1st-gene-specific-NF and 1st-gene-specific-NR, with minimum difference in their Tm values.

    In the example given below, you will see the GC% and Tm value for each forward primer and reverse primer at different over lapped lengths, within the range of 18-30 mer.

    Look for the primer set that contains the minimum Tm difference (∆Tm).  In this example, most Tm values of reverse primer is higher than those of forward primer. Thus, select the reverse primer (22 mer), with lowest Tm (71.4) first. Then select forward primer which has the closest Tm values to the reverse primer’s Tm (71.4) to minimize ΔTm (°C). Therefore, the forward primer (30 mer) with Tm (70.9) is the best choice for 1st-gene-specific-CF.

Sequences of Primers you selected are then shown at “1st-gene-specific-[CN]F” and “1st-gene-specific-[CN]R”.

Please note: For reverse primer, please use the complement sequence at “Complement” field.

Please click here (Right click and choose “save target as” in Internet Explorer or “save link as” in Chrome) to download and save the Primer Design Tool for the 1st PCR.

Please click here (Right click and choose “save target as” in Internet Explorer or “save link as” in Chrome to download the Excel file) to download and save the CFPS700 Primer List.