E.Z.N.A. Plant Direct PCR Kit

Transcription

1 E.Z.N.A. Plant Direct PCR Kit TQ TQ preps 100 preps June 2013

2 E.Z.N.A. Plant Direct PCR Kit Table of Contents Introduction and Overview...2 Kit Contents/Storage and Stability...3 Plant Extraction Protocol...4 PCR Protocol...5 Ordering...7 Manual Revision: June 2013 Innovations in nucleic acid isolation 1

3 Introduction and Overview Introduction The E.Z.N.A. Plant Direct PCR Kit contains all of the reagents required to rapidly extract and amplify genomic DNA from plant leaves. Briefly, the DNA is extracted from a leaf tissue punch ( cm disk) by incubation in an extraction solution at 56 C for minutes and at 95 C for 10 minutes. After an equal volume of PT3 Buffer is added to the extract to neutralize inhibitory substances, the extract is ready for PCR. An aliquot of the diluted extract is combined with the 2X Taq Master Mix and user provided PCR primers to amplify target DNA. The 2X Taq Master Mix is a 2X reaction mix containing buffer, salts, dntps, and Taq DNA Polymerase. It is optimized specifically for use with the extraction reagents. New in this Edition: Proteinase K is now premixed with PT2 Buffer. Proteinase K is no longer included as a separate component in this kit. 2

4 Kit Contents Product TQ TQ Preparations 20 preps 100 preps PT1 Buffer 2 ml 12 ml PT2 Buffer 150 ml 550 ml PT3 Buffer 3 ml 12 ml 2X Taq Master Mix 300 μl 2 x 700 μl User Manual P P Storage and Stability All of the Plant Direct PCR Kit components are guaranteed for at least 12 months from the date of purchase when stored as follows. 2X Taq Master Mix should be stored at -20 C. All remaining components should be stored at 2-8 C.. 3

5 E.Z.N.A. Plant Direct PCR Kit Protocol E.Z.N.A. Plant Direct PCR Kit Protocol - Plant Extraction All steps should be preformed at room temperature unless otherwise noted. Materials and Equipment to be Supplied by User: Incubator capable of 56 C and 95 C 2 ml collection tube 70% ethanol Before Starting: Set incubator to 56 C Set incubator to 95 C 1. Rinse the paper punch and forceps in 70% ethanol prior to use and between different samples. 2. Punch a cm disk of leaf tissue into a 2 ml collection tube or suitable vessel using a standard one hole paper punch. If frozen plant tissue is used, keep the leaves on ice while punching disks. 3. Add 95 μl PT1 Buffer and 5 μl PT2 Buffer to the collection tube. Vortex to mix thoroughly. 4. Incubate at 56 C for minutes. 5. Incubate at 95 C for 10 minutes. 6. Add 100 μl PT3 Buffer. Vortex to mix thoroughly. 7. Store the extraction at 2-8 C. 4

6 E.Z.N.A. Plant Direct PCR Kit Protocol E.Z.N.A. Plant Direct PCR Kit Protocol - PCR Protocol This protocol serves as a guideline for PCR amplification. Optimal reaction conditions, such as incubation times, temperatures, and amount of template DNA, may vary and must be individually determined. Materials and Equipment to be Supplied by User: Thermal cycler Primers DNA Template Molecular-grade water 1. Thaw primer solutions. Keep on ice and mix well before use. 2. Mix the Taq Master Mix by vortexing briefly. It is important to mix the Taq PCR Master Mix before use to avoid localized differences in salt concentration. 3. Prepare one of the following reaction mixes on ice: (For a 25 μl reaction volume) Component Volume Final Concentration 2X Taq Master Mix 12.5 μl 1X Upstream Primer, 10 μm 0.5 μl μm Downstream Primer, 10 μm 0.5 μl μm DNA Template 4 μl < 500 ng Adjust volume to 25 μl with molecular-grade water 4. Gently mix the reaction and spin down in microcentrifuge. 5

7 E.Z.N.A. Plant Direct PCR Kit Protocol 5. Set up a program for a routine PCR reaction: Step Temperature Time Initial Denaturation C 1-5 minutes cycles Denaturation C 30 seconds Anneal C 10 seconds Extension 72 C 1 minute/kb Final Extension 72 C 7 minutes Hold 4-10 C 6. Place the PCR tubes in the thermal cycler and start the cycling program. Optional: For a simplified hot start, begin the PCR program. Once the thermal cycler has reached 94 C, place the PCR tubes in the thermal cycler. In many cases, this simplified hot start improves the specificity of the PCR. 6

8 Ordering Information The following components are available for purchase separately. (Call Toll Free at ) Product E.Z.N.A. SP Plant DNA Kit E.Z.N.A. Plant DNA Kit E.Z.N.A. HP Plant DNA Kit E.Z.N.A. Gel Extraction Kit E.Z.N.A. Cycle Pure Kit Part Number D5511 D3485 D2485 D2500 D6492 HiBind, E.Z.N.A., and MicroElute are registered trademarks of Omega Bio-tek, Inc. PCR is a patented process of Hoffman-La Roche. Use of the PCR process requires a license. 7

9 Notes: 8