How to clone a gene of interest in-frame with a fluorescent protein (alternative protocol)
How to clone a gene of interest in-frame with a fluorescent protein (alternative protocol)
1. Choose any vector carrying a fluorescent protein. Linearize your vector either by restriction digest or inverse PCR.
2. Purify the linearized vector by gel extraction to ensure the isolation of only linear vector molecules.
3. Design In-Fusion PCR primers using the online Primer Design Tool. (The Primer Design Tool is compatible with Mozilla Firefox or Google Chrome web browsers, but not with Internet Explorer.)
-Alternatively, instructions for In-Fusion primer design are described in the In-Fusion Snap Assembly User Manual and the In-Fusion Snap Assembly EcoDry User Manual.
-Regardless of design method, we recommend SnapGene Viewer for in silico assembly of your recombinant construct and easy visualization of the locations of In-Fusion primers in the sense and antisense strands.
-To preserve translational reading frame continuity, your gene of interest should not contain a STOP codon if inserted upstream of the fluorescent protein. If the gene of interest is downstream of the fluorescent protein, it may retain its own STOP codon, but the fluorescent protein should not.
-If necessary, you can also adjust the reading frame continuity by inserting extra nucleotides between the template/gene-specific portion and the 15-nt homologous portion of the In-Fusion PCR primers, as illustrated in the table below. (The current version of our Primer Design Tool does not allow for this type of adjustment, and requires manual design by the user.)
5' 15-nt homology with vector sequence
Number of bases needed to maintain reading frame
3' gene-specific sequence of the In?Fusion PCR primer
4. Amplify your gene of interest using the In-Fusion PCR primers designed in Step 3.
5. Purify the amplified gene of interest using NucleoSpin Gel and PCR Clean-Up or Cloning Enhancer.
6. Follow the In-Fusion Snap Assembly User Manual or the In?Fusion Snap Assembly EcoDry User Manual for In-Fusion reaction and transformation instructions.