View PCR Purification Protocol WEEK 1.pdf from HB 1196 at University of California, San Diego. July 2018 Quick-Start Protocol QIAquick PCR Purification Kit QIAquick PCR & Gel Cleanup Kit The QIAquick Study Resources Product Specifications 6 Introduction 7 The QIAquick Principle 10 QIAquick PCR Purification Kit (50) (250) Catalog no. 28104 28106 QIAquickSpinColumns 50 250 BufferPB* 30ml 150ml Protocol: QIAquick PCR Purification Kit using a Microcentrifuge Th i sp rotc l d eg nu fy -b aDNA mPCR We use the following solutions & protocol to clean up our PCR products prior to cycle sequencing. In general, you will need about 10ng of purified PCR product per 100 bp of length (e.g., for a 500 bp PCR product, you need about 50 ng of template for sequencing). The purpose of this protocol is to remove unused primers and dNTP's from the PCR. Ohio State University The PureLink Quick Gel Extraction and PCR Purification Combo Kit is designed to purify DNA fragments from agarose gels. The simple procedure uses a silica-based spin cartridge to purify DNA fragments from 40 bp to 10kb in <30 minutes from TAE or TBE agarose of various percentages and melting points. The PCR purification protocol achieves rapid non-specific PCR products. • The protocol may be applied for clean-up of PCR products, generated by any thermophilic DNA poly-merase or polymerase mix. • The procedure is not recommended for downstream cloning applications. Reference 1. Werle, E., et al., Convenient single-step, one tube purification of PCR products for direct sequencing, 3.1.4 Heat PCR products with exoSAP in a thermocycler at 37°C for 45 minutes, and then at 80°C for 15 minutes. 3.1.5 Store plates at 4°C until proceeding to the sequencing reaction. 3.2 Thermofisher GeneJET PCR Purification *Notes: This protocol was taken from the user manual of the Thermofisher GeneJET PCR Purification Kit. PCR Purification Kit as described in this manual. The PCR product was analyzed by agarose gel electrophoresis and shows efficient purification of a 965-bp PCR product from a 232-bp PCR fragment and primers. The figure below shows a DNA Ladder (Lane 1), the PCR product before purification (Lane 2), and the PCR product after purification (Lane 3). The amount of PCR product i s usually quantified using a fluorescence detector, and the number of cycles of amplification required PROTOCOL Polymerase chain reaction Usually 20 to 50 μl total in volume and will include the following: X μl, 0.1 to 1 μg of genomic DNA or cDNA, ~0.1μg should be sufficient genomic and fragment/PCR product purification and includes a sample protocol for each type of isolation system. Along with the discussion of Promega's DNA purification systems, we also consider the issues of scalability, downstream applications and yield to assist in finding the best system for your needs. A.Basic Isolation Procedure Protocol to purify PCR products in preparation for cloning using Proteinase K (P8107) Pool up to 400 ul of PCRs, containing 1 ug of the desired product. (If nonspecific products are present in a noticeable amount, gel purify purification is recommended.) Add 1 uL (0.8 U, ~20 ug) Proteinase K. (Proteinase K is active in most common buffers