Do I have to purify the PCR-amplified insert and/or vector prior to performing the In-Fusion Cloning reaction?
Do I have to purify the PCR-amplified insert and/or vector prior to performing the In-Fusion Cloning reaction?
Yes, the PCR-amplified DNA must be purified prior to In-Fusion Cloning. Following PCR, verify by agarose gel electrophoresis that your target fragment has been amplified. 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). However, if nonspecific background or multiple bands are visible on your gel, isolate your target fragment by gel extraction. 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.
1. NucleoSpin Gel and PCR Clean?Up
*Gel extraction enables selection of specific DNA fragments of the desired size from background PCR byproducts or other contaminants.
*Column purification is appropriate if PCR did not produce a background smear.
2. Cloning Enhancer (CE)
*This proprietary enzyme mix removes background plasmid DNA and PCR residue.
*CE is appropriate for PCR that results in a single fragment of the expected size, without a background smear.
*CE is a convenient tool for HTP applications that employ highly optimized PCR cycling conditions and primers such that PCR generates clean DNA fragments of the expected size.
Note: In most cases, CE treatment does not require additional column purification or gel extraction. However, to ensure better cloning results, PCR-linearized vectors may require a combination of CE treatment followed by gel extraction to separate a linearized vector from possible PCR byproducts.