Download BiotoolTM Plant Leaf Direct PCR Kit (B4003)

BiotoolTM Plant Leaf Direct PCR Kit (B4003)
Introduction
The BiotoolTM Plant Leaf Direct PCR Kit provides a fast one-step preparation and PCR amplification that is specifically designed for multiple plants
excluding the polysaccharide and polyphenols plants. Biotool’s trademarked Buffers rapidly digest plant leaves to release intact genomic DNA
that can be used directly as template for PCR amplification. By using this kit, the genomic DNA extractions can be done within 10-15 minutes. In
addition, the Biotool 2x P-PCR OPTITM Mix (which includes an optimized Taq Polymerase) ensures high amplification efficiency of target DNA.
Buffer B
10 minutes
PCR
95 oC
2x P-PCR
OPTITM Mix
Buffer A
Application
Direct PCR of plant leaves including rice, wheat, maize, rapeseed plant, soybean and other plants.
The BiotoolTM Plant Leaf Direct PCR plus Kit is available for polysaccharide and polyphenols plants including arabidopsis, cotton, banana, potato,
tomato, etc.
Kit Contents
Catalog #
B40023 (200 rxns)
B40025 (500 rxns)
B40028 (2000 rxns)
Buffer A (mL)
10
25
100
Buffer B (mL)
10
25
100
2x P-PCR OPTITM Mix (mL)
2
5
20
6x DNA Loading Buffer (mL)
0.6
1.5
6
User Guide
Yes
Yes
Yes
Contents
Buffer A: Lysis buffer
Buffer B: Neutralization buffer
2x P-PCR OPTITM Mix: Includes Biotool’s trademarked and optimized Taq DNA polymerase, dNTPs, MgCl2, and reaction buffer.
6x DNA Loading Buffer: We strongly recommended this loading buffer in agarose gel electrophoresis.
Storage
2x P-PCR OPTITM Mix should be stored at -20 ℃. If the mix is to be used frequently, it can be stored at 4 ℃ for up to 10 days.
Buffers can be stored at 4 ℃ for up to 2 years.
Experimental Protocol
1. Genomic DNA Preparation
Place plant leaf sample (5-10 mg, diameter 5-7 mm) in a centrifuge tube. Add 50μL of Buffer A (be sure to submerge the entire sample). Incubate
the tube at 95 ℃ for 10 minutes. Add 50μL of Buffer B and vortex briefly.
2. PCR
Add primers and template into 2x P-PCR OPTITM Mix according to the recommended concentrations. Give the mixture a quick spin in the
centrifuge and load into PCR amplifier to begin amplification.
PCR Reaction Components
20 μL Reaction Volume (μL)
50 μL Reaction Volume (μL)
2x P-PCR OPTI
10
25
Forward Primer (10 μM)
0.5
1
Reverse Primer (10 μM)
0.5
1
Template
2
5
H2O
7
18
Total Reaction Volume
20
50
Temperature (℃)
Time
Cycles
94
5 min
1
94
20 sec
45-65
30 sec
72
X min (1kb/min)
72
5 min
1
12
--
1
TM
Mix
30-40
Note: Before First Use
Avoid repeat freeze-thaw cycles of the 2x P-PCR OPTITM Mix. Please note that if the mixture turns viscous (especially at high temperature),
simply incubate on ice for 1-2 minutes, and then gently shake 3-5 times prior to using.
Trouble Shooting
Problem
No amplification
product in test or
control samples
Amplification worked
in the control samples,
but not in test samples
Non-specific
amplification product (s)
Over-exposed signal
on agarose gel
Potential Cause(s)
Suggestion(s)
Amplification reaction was incorrectly set up
Optimize the proper reaction set up
Improper storage has led to loss of activity of PCR reagents
Replace all components with fresh reagents
Primers are not optimal and did not anneal
Redesign primers
Digestion was incomplete
Extend digestion time up to 20 minutes at 95℃
Lysis solution was stored too long and genomic DNA degraded
Collect fresh plant leaf samples for genomic DNA extraction
Unknown contaminants interfered with amplification reaction
Dilute template in purified H2O or TE buffer and repeat PCR
The quantity of the amplification product was not sufficient
Increase the number of PCR cycles to 35-40 to yield more
amplification product
Annealing temperature too low
Increase the annealing temperature
The number of PCR cycles was too high
Decrease the number of cycles to 30-35
Primer concentration was too high
Decrease primer concentration
Template concentration was too high
Dilute template in purified H2O or TE buffer
Loading dye may be sub-optimal for your specific reaction product
Switch to a different brand of loading dye