E.Z.N.A.® Plant Direct PCR Kit
TQ2800-00
20 preps
TQ2800-01
100 preps
June 2013
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
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 (0.5-0.7cm disk) by incubation in an extraction solution at 56°C for 10-30
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:
•
•
2
Proteinase K is now premixed with PT2 Buffer.
Proteinase K is no longer included as a separate component in this kit.
Kit Contents
Product
TQ2800-00
TQ2800-01
20 preps
100 preps
PT1 Buffer
2 mL
12 mL
PT2 Buffer
150 mL
550 mL
PT3 Buffer
3 mL
12 mL
300 μL
2 x 700 μL
P
P
Preparations
2X Taq Master Mix
User Manual
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
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 0.5-0.7 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 10-30 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
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
0.1-1.0 μM
Downstream Primer, 10 μM
0.5 μL
0.1-1.0 μM
4 μL
< 500 ng
DNA Template
Adjust volume to 25 μL with molecular-grade water
4.
Gently mix the reaction and spin down in microcentrifuge.
5
E.Z.N.A.® Plant Direct PCR Kit Protocol
5.
Set up a program for a routine PCR reaction:
Step
Initial Denaturation
Temperature
Time
94-95°C
1-5 minutes
25-40 cycles
Denaturation
94-95°C
30 seconds
Anneal
45-70°C
10 seconds
Extension
72°C
1 minute/kb
Final Extension
72°C
7 minutes
4-10°C
∞
Hold
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
Ordering Information
The following components are available for purchase separately.
(Call Toll Free at 1-800-832-8896)
Product
Part Number
E.Z.N.A.® SP Plant DNA Kit
D5511
E.Z.N.A.® Plant DNA Kit
D3485
E.Z.N.A.® HP Plant DNA Kit
D2485
E.Z.N.A.® Gel Extraction Kit
D2500
E.Z.N.A.® Cycle Pure Kit
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
Notes:
8