Bio-Medical Department Data sheet 80XDNA-Plant-602

PI-80X Plant DNA 1 protocol

Genomic DNA isolation from rice leaves
- automation from plant cell lysis to DNA dissolution -

KURABO PI-80X is an automated DNA isolation system. This data sheet shows an example of
DNA isolation from rice leaves with Plant DNA1 protocol. With options such as heater and
additional dispensing lines, PI-80X processes from cell lysis to dissolution of DNA automatically.

- Liquid nitrogen
- Zirconic beads

PI-80X with heater and additional

Grind with tissue dispensing lines (option)
homogenizer SH-48
Specially designed Plant DNA1 protocol
8-hole tube (PT-8000)

Experiment
Sample Rice leaves (Oryza sativa)
Sample amount 100mg
Isolation system and PI-80X with heater and additional dispensing lines (options)
protocol Plant DNA1 protocol

Reagents kit NR-501

and consumables Sample tube, PT-8000; DNA tube, NT-8000
Chemical principle Lysis of cells: CTAB and detergent
Purification: Chloroform and salt
DNA isolation: Precipitation by alcohol
Process Pre-treatment:
Freeze rice leaves in liquid nitrogen in 8-hole tube (KURABO, PT-8000), and
grind with the SH-48 tissue homogenizer (KURABO).

DNA isolation with PI-80X with heater and additional dispensing

lines (options):
Step1. Lysis reaction at 65C for 40min
Step2. Denature protein and remove debris
Step3. Precipitate DNA
Step4. Wash DNA
Step5. Dry up
Step6. Dissolve DNA in DNA dissolving reagent (KURABO, PR6025)
Final volume 100ul

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Bio-Medical Department Data sheet 80XDNA-Plant-602

Workflow of Plant DNA protocol

Sample tube
Reagent7 Reagent1 Reagent2 Reagent3 Transfer supernatant
(Decantation)

Heat at 65C Agitation Agitation Agitation Centrifugation

with optional heater

Lysis of cell Denature protein and remove debris

DNA tube
Waste supernatant Reagent5 Waste supernatant
Reagent8 Waste supernatant
Reagent9 Reagent4 Reagent6
(Decantation) (Decantation)
(Decantation)

Agitation Centrifugation Agitation Agitation Centrifugation Agitation Centrifugation Agitation

Remove polysaccharide Precipitate DNA Wash DNA Dry-up Dissolve DNA

(Plant DNA2 protocol)

Processing time 48 samples: 2.8hr

(PI-80X) 48 samples: 2.1hr with optional heater unit

Analysis methods
Yield and purity Absorbance of 260nm was measured for each DNA solution by spectrometer.
calculation DNA yield was calculated as follows:
A260 X 50 X dilution factor X final volume.
DNA purity was evaluated by the ratio of A260/A280.

Electrophoresis Each 5µl of DNA solution was loaded on a 0.7% agarose gel.

Restriction enzyme Each 1ug of DNA isolated from rice leaves was digested with 20units of enzymes for
digestion 15hours at the optimal temperature, respectively.
Restriction enzyme: EcoRI and BamHI

PCR amplification Template: 100ng of isolated DNA from rice leaves

Target gene: Ribrose-1,5-bisphosphate carboxylase / oxygenase large subunit gene,
amplicon size 670bp
Polymerase: AmpliTaq® DNA Polymerase (0.6U)
PCR condition: 94C, 5min x 1 cycle
94C, 20sec/60C, 40sec/72C 1.5min x 35 cycles
72C, 7min x 1 cycle
Reaction volume: 25ul
Electrophoresis: 10ul of 25ul reaction mixture was loaded on a 1.8% agarose gel.

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Bio-Medical Department Data sheet 80XDNA-Plant-602

Results
Yield and purity Yield (ug) Purity (A260/A280) Purity (A230/A260)
21/100mg 1.79 0.46

Electrophoresis
M
Each 5ul of 100ul DNAsolution
isolated from rice leaves
M: λHindIII size maker

Restriction enzyme Each 1ug of DNA isolated from mouse tail was digested
M 1 2 3 4 5 6
digestion with restriction enzymes.
M: λHindIII size maker
1: DNA isolated with manual kit in market (undigested)
2: Manual kit, EcoRI digested
3: Manual kit, BamHI
4: DNA isolated with PI-80X (undigested)
5: PI-80X, EcoRI
6: PI-80X, BamHI

PCR amplification M1 C- C+ 1 2 3 4 5 6 7 8 M2
M1: φX174 HinII size marker
C-: Negative contol
C+: Positive contol
1-8: Amplicon of isolated DNA
M2: φX174 HinII size marker

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Bio-Medical Department Data sheet 80XDNA-Plant-602

Ordering information
DNA isolation system PI-80X
Option: Heater and additional dispensing lines
Plant DNA1 protocol

Reagents kit Plant DNA isolation reagents kit ver.1 NR-501 (approx. for 700 preps)
Reagent name Reagent No. (Code) Content
Denaturing reagnet A 1 (SR1050) 1
Denaturing reagent B 2 (SR2050) 1
Deproteinization reagnet 3 (SR3025) 1
Precipitating reagent 4 (PR4050) 1
Washing reagent 5 (PR5050) 4
DNA dissolving reagent 6 (PR6025) 1
Cell lysing reagent 7 (SR7050) 1

Processing tube Sample tube: PT-8000 (8-hole, blue, 64pieces)

DNA tube: NT-8000 (8-hole, white, 64pieces)

KURABO INDUSTRIES LTD.

Bio-Medical Department
4-31, 2-chome, Kyutaro-machi, Chuo-ku
Osaka 541-8581, Japan
Phone: 81-6-6266-5010
Fax: 81-6-6266-5011
bio@ad.kurabo.co.jp

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