CE is appropriate for PCR that results in a single fragment of the expected size, without a background smear.This proprietary enzyme mix removes background plasmid DNA and PCR residue.Column purification is appropriate if PCR did not produce a background smear.Gel extraction enables selection of specific DNA fragments of the desired size from background PCR byproducts or other contaminants.If you use PCR to amplify your vector and insert and you obtain both a PCR-amplified vector and PCR-amplified fragment(s) without nonspecific background, you can use the Quick In‑Fusion Cloning Protocol provided in Appendix A of the In-Fusion HD Cloning Kit User Manual. However, if nonspecific background or multiple bands are visible on your gel, isolate your target fragment by gel extraction. If a single band of the desired size is obtained, you can either spin-column purify ( NucleoSpin Gel and PCR Clean‑Up) or treat your PCR product with Cloning Enhancer (CE). Following PCR, verify by agarose gel electrophoresis that your target fragment has been amplified. Yes, the PCR-amplified DNA must be purified prior to In‑Fusion Cloning. How do I clone my gene of interest in the same translational reading frame as a tag present in the cloning vector (e.g., fluorescent protein, Myc, HA, etc.)? Number of bases needed to maintain reading frameģ' gene-specific sequence of the In‑Fusion PCR primer For example: 5' 15-nt homology with vector sequence To shift the reading frame, you would simply add one or two additional bases after the 15-bp homology and before the first codon of the target gene. For example, when creating a fusion protein, if the 15 bp of vector homology at the 5' end of the In‑Fusion PCR primer sequence corresponds to the last five codons of the vector reading frame, you would clone your new gene or tag in the same reading frame downstream of the C-terminus of the vector sequence by placing the first codon of this gene next to the last codon of the homology sequence (i.e., at the 3' end) without any interfering STOP codons. The reading frame is defined by the primer sequence. What is the optimal length of the homologous overlap between the termini of the PCR-amplified insert and linearized cloning vector? (Optional) To ensure continuity of the translational reading frame, or to preserve restriction site(s), additional nucleotides can be added to the PCR primer(s) between the template-specific portion and the 15-nt homologous overlap. The 15-bp complementary regions must be located at the termini of adjacent DNA fragments or they will not be joined by In‑Fusion Cloning. Homologous overlaps shorter than 12 nt and longer than 21 nt are not recommended. For multiple-insert cloning, we recommend increasing the homology to 20 nt.
0 Comments
Leave a Reply. |