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Polymerases

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EZ™ Plant Direct PCR Kit

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1 EZ™ Plant Direct PCR Kit (50 µl reactions) P810 2X 200 reactions ₩ 270,000 추가
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Product description

EZ™ Plant Direct PCR Kit was developed to be able to perform the PCR immediately without DNA purification process from the leaves of the plant. The 2X Plant PCR MIX, which includes loading dye HOT-Start DNA Polymerase, and reaction buffer of the components of this Kit, it is possible to reduce the errors in the course of the experiment, it can be used conveniently. It has also been developed to have a high resistance to PCR inhibitor their various present in plants. Control primer that can be used to amplify the conserved region of the chloroplast DNA is included in this Kit.

 

Kit components

- Dilution buffer

- 2X Plant PCR MIX

- Control Primer MIX (10 pmol/µl each)

- Sterile water (RNase free)

 

Standard Direct PCR protocols

1. Please experimenting of glove wear certainly after to prevent RNase contamination

2. Thawing and opening of the product: Please be thawed in lap-top cooler or on Ice.

Reagents that have been thawed, after the spin-down, please use by uniformly mixing.

3. Sample preparation from the plant leaves: recommend that you use the young leaves. 

 

Sample Handling

1. Cut vertically and horizontally than 1 mm leaves of the plant.

* TIP

1) recommend a 0.35 mm sampling tool or Harris Uni-Core™ 0.5 mm.

2) You may also be used in well washed Carter blade or razor blade.

3) It does not have to be prepared to take lightly the blade to 100 μl pipette tip and tip 20 μl.

2. After turning the leaves of the plants which had been prepared in to put Buffer 20 μl Dilution to

1.5 ml EP tube, and to squish 6-8 times to be a green Dilution Buffer is thin to 100 μl pipette tip.

After centrifugation, is used as a template in 50 μl PCR reaction by taking the 0.5 μl supernatant.

* Note

1) The required amount of supernatant, may vary depending on the type of plant.

2) Please use it to increase to 50 or more μl the amount of Dilution buffer if the amount of plant

leaves in many cases.

3) If you want to try Direct PCR for the first time, the task of adjusting the amount of sample

amount Dilution buffer in accordance with the conditions of use is essential. 

 

PCR mixture (standard 50 µl reaction)

2X Plant PCR MIX

25 μl

Forward and Reverse Primer (10 pmol)

μl

Diluted sample

0.5 μl

RNase free water

up to 50 μl

 

PCR cycles

Step

Temperature

Time

Cycle

Initial denaturationa

95

10 min

1

Denaturation

95

30 sec

30~40

Annealingb

55~65

30~60 sec

Elongation

72

1 min/kb

Final elongation

72

5 min

1

aAt least 10 min of initial denaturation time is required to fully activate the chemically modified PCR DNA polymerase

bRecommended annealing temperatures is 5 to 10 below the lower Tm of the two primers used.

 

Troubleshooting

If there is any Product, if yield is low

Make sure that the Direct PCR control reaction using the control primer and Purified DNA PCR is

working.

And template0.5 μl using PCR at a dilution of 1:10 or 1:100 in DW supernatant from the sample was prepared using Dilution buffer.

Either decrease the Sample size, and increasing the amount of Dilution buffer.

If Nonspecific product occurs

Include a negative control using by washing with 2% sodium hypochlorite and Cutting tool.

If it is smearing upwards, to reduce the amount of primer.

Reduce the cycle number when the product of the size of the other results, to design a new primer.

 

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