CloneID 1X Colony PCR Master Mix FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE. Lucigen Corporation 2905 Parmenter St, Middleton, WI 53562 USA Toll Free: (888) 575-9695 (608) 831-9011 FAX: (608) 831-9012 lucigen@lucigen.com www.lucigen.com
Table of Contents Components & Storage Conditions... 3 Description... 3 Introduction to PCR... 3 PCR Setup... 4 PCR Cycling Conditions... 4 PCR Guidelines... 5 Quality Control... 6 Technical Support Lucigen is dedicated to the success and satisfaction of our customers. Our products are tested to assure they perform as specified when used according to our recommendations. It is imperative that the reagents supplied by the user are of the highest quality. Please follow the instructions carefully and contact our technical service representatives if additional information is necessary. We encourage you to contact us with your comments regarding the performance of our products in your applications. Thank you. Lucigen Technical Support Email: techsupport@lucigen.com Phone: (888) 575-9695 Product Guarantee: Lucigen guarantees that this product will perform as specified for one year from the date of shipment. Please avoid using reagents for greater than one year from receipt. 2
Components & Storage Conditions Components: CloneID 1X Colony PCR Master Mix contains all of the components necessary to perform PCR amplification, except for template and primers. Catalog # Number of reactions 30059-0 (Trial Size) 50 reactions (1 tube containing 1250 µl) 30059-1 250 reactions (5 tubes, each containing 1,250 µl) 30059-2 500 reactions (10 tubes, each containing 1,250 µl) Store at -20 C Storage: CloneID 1X Colony PCR Master Mix can be stored at -20 C for 12 months. The product needs to be mixed well prior to use. It is stable for ten freeze-thaw cycles. Description CloneID 1X Colony PCR Master Mix is simple and reliable solution for Colony PCR. Simply add your primers and a small amount of a colony from a plate, then cycle the reaction. No need to add water, optimize Magnesium concentration, or lyse cells. This master mix is robust under challenging conditions, such as genotyping directly from cells, so you get the most reproducible and reliable results every time. The product is also formulated with green gel loading dye for easy gel loading and visualization. It s the fastest and easiest Colony PCR product available. All necessary reaction components are provided in the 1X Master Mix, which contains the following as provided: CloneID DNA Polymerase, Reaction Buffer (ph 8.8, 40 mm Tris), 220 µm datp, 220 µm dgtp, 220 µm dctp, 220 µm datp, 2.2 mm MgCl 2, 11 mm KCl, 11 mm (NH 4) 2SO 4, 0.11% Triton X- 100, and a proprietary mix of PCR Enhancer/Stabilizer. PCR using CloneID 1X Colony PCR Master Mix should undergo denaturation at 98 C for best results. This product is primarily intended for use with E. coli colonies and DNA samples. CloneID has not been tested for diverse organisms. Introduction to CloneID PCR The Polymerase Chain Reaction (PCR) is a powerful technique that amplifies specific DNA sequences using multiple cycles of a 3-step process. The first step involves denaturation of double-stranded DNA templates at the elevated temperature of 98 C. In the second step, sequence-specific primers anneal to complementary sites flanking the target sequence. In the third step, thermostable CloneID DNA polymerase extends the annealed primers, copying the original DNA sequence. The newly synthesized DNA becomes the template for subsequent DNA amplification, doubling the amount of template with each cycle. These steps are repeated 25-35 times, resulting in a 10 5-10 9 fold increase in the amount of target DNA. Performing up to 45 cycles can improve sensitivity but may lead to increased non-specific amplification. 3
PCR Setup Colony PCR: Colony PCR is a method in which live cells are taken from a colony on a plate and added directly to the PCR reaction without lysis or sample prep of any kind. During the initial denaturation step the cells are lysed, immediately providing template DNA for the reaction. CloneID is specifically designed specifically to excel at this method. CloneID is a 1X master mix, so no water needs to be added. However, you may alternatively add up to 5 µl of template DNA to the reaction instead of colony matter. Reaction setup for either method is described below. See the PCR Guidelines sections for more information. 1) Materials supplied by the user. PCR amplification is performed by adding template (purified DNA or whole cells) and primers to the 1X CloneID Master Mix. The following components must be supplied by the user: Fresh colonies, or Template DNA (10-50 ng plasmid DNA or 10-200 ng genomic DNA, in <5 µl) Forward Primer (50-100 µm) Reverse Primer (50-100 µm) Thermocycling apparatus Pipet tip, sterile loop or toothpick for picking colonies 2) Reaction Setup. Set up PCR amplifications of the desired size, according to the following chart. 25 µl Reaction* 50 µl Reaction 100 µl Reaction Final Concentration CloneID 1X Master Mix 25.0 µl 50 µl 100.0 µl 1 X Forward Primer (50 µm) 0.25 µl 0.5 µl 1.0 µl 0.5 µm Reverse Primer (50 µm) 0.25 µl 0.5 µl 1.0 µl 0.5 µm DNA template** 1 portion of a colony or 1.0 µl of DNA 1 portion of a colony or 1.0 µl of DNA 1 portion of a colony or 1.0 µl of DNA Please see PCR Guidelines below for information on picking colonies for PCR. * Standard recommended reaction size is 25 µl. ** If adding a solution of DNA, we recommend adding 1 µl at 10 ng/µl. For best results, add no more than 5 µl total to the reaction (including primer volume). 3) Gently mix the PCR components in a thin-walled reaction tube and spin briefly in a microcentrifuge. Add a drop of mineral oil if the thermocycler does not have a heated lid. 4
PCR Cycling Conditions 1) Pre-heat the thermocycler to 98 C. 2) For initial cell lysis of colonies and denaturation of target template, incubate the reactions at 98 C for 2 minutes. 3) Denature, anneal, and extend the DNA according to the cycling conditions (next page): Cycling step Temperature Time # of Cycles Lysis / Denaturation* 98 C 2 min 1 Denaturation* 98 C 15-30 sec Annealing** 50 65 C 15-30 sec Extension 72 C 1 min/kb 25 45 Final Extension 72 C 5-10 min 1 Hold 4 C Indefinitely 1 * CloneID Colony PCR reactions must undergo denaturation at 98 C for optimal results. **Anneal at primer melting temperature (T m) ±2 C. See PCR Guidelines section. 4) After completion of the PCR, the Colony PCR GREEN reactions can be loaded directly onto an agarose gel for analysis (e.g. 5 µl directly onto a 0.7-2% agarose gel). The Master Mix contains blue and yellow tracking dyes that will separate upon electrophoresis. CloneID PCR Guidelines Optimization of PCR conditions may be required for amplification of templates with high GC content, internal secondary structure, or products greater than 5 kb. The following guidelines can be used: 1) Reaction Set-Up For best results set up and maintain CloneID Colony PCR reactions on ice. There is no hot-start functionality of this product. 2) Template DNA. For colony PCR, a small portion of a colony from a plate is used to provide the template DNA. Using a sterile pipette tip, simply touch a single colony on a plate and add the adherent colony material to the PCR reaction directly. It is not necessary to take an entire colony. Gently pipette up and down a few times to mix the material, being careful not to create bubbles. a. If using purified DNA instead of a colony, we recommend using 10 pg - 50 ng of plasmid DNA and 10-200 ng of genomic DNA. Template should be dissolved in water, rather than EDTA-containing solution such as TE buffer. The amount of template DNA required is dependent on several factors, including the size and complexity of the template DNA and the number of PCR cycles. b. CloneID is primarily intended for use with E. coli colonies and DNA samples. CloneID has not been tested for diverse organisms. 5
3) Primer Design. Oligonucleotide primers for PCR are generally 20-25 bases in length and have a GC content of 40-60%, with the GC bases evenly spaced in the primer. Selfannealing of primers leads to production of primer-dimers, which can diminish the amount of authentic product. The 3 end of each primer should not be complementary to any portion of either itself or the opposing primer. The melting temperature of the primers should be within 5 C of each other. The final concentration of each primer in the reaction should be 0.2-1.0 µm 4) Annealing. CloneID contains PCR enhancers that may lower primer T m slightly. Set the annealing temperature to 2 C below the calculated melting temperature of the primers. If necessary, optimize the annealing temperature for the primer/template combination. Reducing the annealing temperature improves yield and reaction efficiency, while increasing annealing temperature improves specificity. 5) Denaturation Temperature. CloneID master mix has been designed to undergo denaturation at 98 C, both during initial denaturation and with each cycle. To ensure maximum cell lysis and to amplify the very toughest templates, please set all denaturation steps to 98 C. Quality Control PCR Activity: CloneID 1X Colony PCR Master Mix is tested in DNA amplification using a variety of templates and primers, including DNA directly from E. coli colonies. Activity Determination: One unit of enzyme catalyzes the incorporation of 10 nmoles of dntp into acid-insoluble material in 10 minutes at 70 C in 10 mm Tris-HCl (ph 9.0), 50 mm KCl, 1.5 mm MgCl2, 200 µm dgtp, datp, dttp, dctp (a mix of unlabeled and [33P]dCTP), 10 µg Activated Calf Thymus DNA, 0.1% Triton X-100 and 0.1 mg/ml BSA. The master mix is formulated at 0.07 U/µL Absence of Endonuclease or Nicking Activity: Incubation of 10 U of DNA Polymerase with 1 µg of supercoiled puc19 DNA for 16 hours at 37 C results in no detectable conversion to relaxed or linear forms detectable by agarose gel electrophoresis. Purity: CloneID DNA Polymerase is >95% pure as determined by SDS-PAGE. There is no detectable DNA contamination. Notice of Limited Label License, Copyright, Patents, Warranties, Disclaimers, and Trademarks A complete list of trademarks, registered trademarks, limited label license, etc. held by Lucigen Corp. can be found at http://www.lucigen.com/legal-information.html 6