Automated DNA isolation system GENE PREP STAR. March 16th, 2010 KURABO INDUSTRIES LTD. 1
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1 Automated DNA isolation system GENE PREP STAR PI-80X March 16th, 2010 KURABO INDUSTRIES LTD. 1
2 GENE PREP STAR PI-80X is a fully automated and flexible DNA isolation system. Key features Ease-of-use Built-in centrifuge and specially designed tube unit eliminates transfer liquid culture or reaction mixture. Parameter selection on the touch screen panel Safe and sturdy Interlock system of front door System check before starting isolation process Flexible Parameter settings Optional add-ons to expand capacity and usefulness Tube supplying unit Heater unit Reagent dispensing lines March 16th, 2010 KURABO INDUSTRIES LTD. 2
3 Fully automated process from collection cells to dissolution of plasmid DNA 1. Picking up colonies 2. Cultivation 3. Set and run KURABO 8-hole tube unit Touch screen panel Select a protocol to run Select a parameter setting file System check START March 16th, 2010 KURABO INDUSTRIES LTD. 3
4 Fully automated process from lysis reaction to genomic DNA 1. Cut off animal tissue 2. Set and run KURABO 8-hole tube unit 1. Grinding plant material Heater unit (option) keeps reaction temperature. You have only to put sample into tube units. March 16th, 2010 KURABO INDUSTRIES LTD. 4
5 PI-80X isolates 48 of plasmid DNA or genomic DNA per run. Protocol Plasmid DNA Tissue DNA Sample volume Liquid culture 0.5-3ml* Up to 30mg Sample E. coli plasmid/bac Mouse tissue Plant DNA 1 Up to 300mg Arabidopsis Plant DNA 2 Up to 100mg Rice leaves *Optimum culture volume depends on strain of E coli, type of plasmid and culture conditions. March 16th, 2010 KURABO INDUSTRIES LTD. 5
6 Protocols and their processing step Plasmid Tissue DNA Plant DNA 1 Plant DNA 2 Sample source Sample Bacterial culture Liquid culture Animal tissue Cell culture Mouse tail Arabidopsis Leaves Rice Leaves, Sample preparation Process Not required. Concentrate cell Lyse cell Remove debris Precipitate DNA Wash DNA Protease K digestion Denature protein Remove debris Precipitate DNA Wash DNA Dry up Grind and CTAB digestion Lyse cell Remove debris Precipitate DNA Wash DNA Dry up Grind and CTAB digestion Lyse cell Remove debris Remove polysaccharide Precipitate DNA Wash DNA Dry up Elute DNA Elute DNA Dry up Elute DNA Elute DNA March 16th, 2010 KURABO INDUSTRIES LTD. 6
7 Safety and functionality in stylish design Dispensing nozzle Robot Touch screen panel Agitator Front door Interlock Centrifuge Tube rack Moving rack table Waste flood alert Waste fluid bottle tray 6-Channel dispenser March 16th, 2010 KURABO INDUSTRIES LTD. 7
8 Flexible on parameter setting Customization of protocol files Centrifugation speed and time, dry up time, reagent volumes dispense, and so on. 8 protocol files can be saved. For example, 1 for default plasmid, 1 for customized plasmid, 1 for default tissue DNA Protocol files are saved in a CF card. March 16th, 2010 KURABO INDUSTRIES LTD. 8
9 Optional add-ons expand PI-80X s capacity and functionality. Sample Stacker To increase sample number of in one run. From 48 samples/run to 192 samples/run Heater unit To shorten DNA dry up time. For fully automation of genomic DNA isolation from lysis reaction to DNA dissolution. Additive dispensing lines To save trouble to change reagents bottles before running another protocol than the last one. March 16th, 2010 KURABO INDUSTRIES LTD. 9
10 Sample stacker enables PI-80X to process up to 192 samples per run. March 16th, 2010 KURABO INDUSTRIES LTD. 10
11 Heater unit shortens processing time and achieves fully automated gdna preparation from tissue sample to gdna. Agitation under temperature control Airtight covering Infrared ceramic heater March 16th, 2010 KURABO INDUSTRIES LTD. 11
12 Protocols and their processing step with heater unit Plasmid Tissue DNA Plant DNA 1 Plant DNA 2 Sample source Sample Bacterial culture Liquid culture Animal tissue Cell culture Mouse tail Arabidopsis Leaves Rice Leaves, Sample preparation Not required. Not required Grind is SH48 Grind in SH48 Process Concentrate cell Lyse cell Remove debris Precipitate DNA Wash DNA Dry up Protease K digestion Denature protein Remove debris Precipitate DNA Wash DNA Dry up CTAB digestion Lyse cell Remove debris Precipitate DNA Wash DNA Dry up CTAB digestion Lyse cell Remove debris Remove polysaccharide Precipitate DNA Wash DNA Elute DNA Elute DNA Elute DNA Dry up Elute DNA March 16th, 2010 KURABO INDUSTRIES LTD. 12
13 Heater unit shortens time for dry up of DNA. Processing time of plasmid protocol at default parameter setting with or without optional heater unit. Number of samples * Process time from start to dry up of DNA 160 >> 120 min 650 >> 490 min Process time from start to dissolution of DNA 170 >> 130 min 690 >> 530 min * With optional sample stacker March 16th, 2010 KURABO INDUSTRIES LTD. 13
14 Heater unit achieves fully automation from tissue digestion to DNA. DNA isolation Cut off mouse tail Protease K digestion Genomic DNA Cut off mouse tail Protease K digestion and DNA isolation Genomic DNA March 16th, 2010 KURABO INDUSTRIES LTD. 14
15 Additive dispensing lines save trouble to change reagent bottles. 3 or 6 of lines can be added. Example Two protocols are installed in one machine. Just with standard 6 dispensers Plasmid protocol changing the reagent bottles Tissue DNA protocol With option Plasmid: 6 lines (standard) + Tissue DNA: 6 lines (option) Standard 6 lines Optional 6 lines March 16th, 2010 KURABO INDUSTRIES LTD. 15
16 Number of dispensing lines required for protocols combined with lysis reaction Protocol Plasmid Tissue DNA Plant DNA 1 Plant DNA 2 Plasmid + Tissue DNA Number of dispensing lines (Standard: 6) Without lysis of tissue With lysis of tissue* Plasmid + Plant DNA 1 Plasmid + Plant DNA * Optional heater unit is required to do lysis in PI-80X March 16th, 2010 KURABO INDUSTRIES LTD. 16
17 Example 1. Plasmid isolation from E. coli liquid culture. Gel electrophoresis analysis Plasmid: puc18/e. coli JM109 Sample volume: 2ml or 3 ml of LB liquid medium. Isolation condition: PI-80X plasmid DNA protocol (default). Dissolution volume was 100ml. Gel electrophoresis: 0.7% agarose gel. 5µl of DNA solution was loaded in each well. Yield 8µg from 2ml of liquid culture of E. coli JM109 containing puc18 (high copy plasmid). From 3ml culture From 2ml culture Restriction enzyme digestion Plasmid DNA: puc18 isolated with PI-80X plasmid protocol with default parameters. Digestion condition: Each 3units of enzyme showed in the picture below digested 300ng of puc18 for 1 hour at its optical temperature. Each enzyme digests puc18 only at one site, generating one fragment. EcoRI BamH I KpnI ScaI HincI I SmaI March 16th, 2010 KURABO INDUSTRIES LTD. 17
18 Example 1. (continued) Plasmid isolation from E. coli liquid culture. Sequencing analysis Template DNA: 300ng of plasmid puc18 Assay kit: ABI PRISMR Big Dye Terminator Cycle Sequence Ready Reaction Kit (Ver.1.1) Primer: -21M13 System: ABI PRISM 310 Comparison of confidence with manual kit Method Base PI-80X 100% 99.7% 99.4% 98.7% 97.7% Company A manual kit 100% 100% 99.5% 99.1% 98.1% March 16th, 2010 KURABO INDUSTRIES LTD. 18
19 Example 2. Genomic DNA isolation from mouse tissues Gel electrophoresis analysis Sample: 10mg of tail and, 20mg of brain, liver, kidney and heart Pre-treatment: Digestion reaction mixture containing 0.2mg/ml of protease K. At 55 degrees of C for 15 hours. Isolation condition: PI-80X Tissue DNA (default) Dissolution volume: 100µl of TE buffer. Electrophoresis: 0.7% agarose gel, 5µl of DNA solution loaded Tail Brain Liver Kidney Heart Yield Tissue Attributes Yield (µg/mg tissue)* Purity (A230/A260) Tail Brain Liver Kidney Heart * The processed amount was 10mg for tail, 20mg for the other tissues. Sample volume Mouse tail: up to 10mg (5-10mm) Other tissues: up to 30mg March 16th, 2010 KURABO INDUSTRIES LTD. 19
20 Example 2. (continued) Genomic DNA isolation from mouse tissues PCR analysis Template: 200ng Target gene: Mouse ICAM 1, exon 6-7 Amplicon size: 535bp Polymerase: AmpliTaq Gold DNA Polymerase (0.6U) PCR condition: 94C, 5min x 1 cycle 94C, 20sec/68C, 40sec/72C 1.5min x 35 cycles 72C, 7min x 1 cycle Reaction volume: 25µl Electrophoresis: 10µl of 25µl reaction mixture was loaded on a 1.8% agarose gel. M C+ C M: φx174 Hin II size marker C+: Positive control C-: Negative control 1-7: Amplicin of gdna sample isolated with PI-80X Restriction enzyme digestion Reaction condition: 10units of enzyme digested 1mg of genomic DNA from mouse tail at optical temperature for 55 hours. M C M: λhindⅢ size marker C: undigested DNA 1: EcoRI 2: KpnI 3: Sac I 4: BamHI March 16th, 2010 KURABO INDUSTRIES LTD. 20
21 Example 3. Genomic DNA isolation from plant material Gel electrophoresis analysis Sample: 100mg of rice leaves Pre-treatment: Frozen with liquid nitrogen and ground with tissue grinder SH-48 (KURABO) Isolation condition: PI-80X Plant DNA 2 (default) Dissolution volume: 100µl of TE buffer Electrophoresis: 0.7% agarose gel, 5µl of DNA solution loaded M M: λhindiii size marker 1-8: Genomic DNA isolated with PI-80X Sample volume Arabidopsis leaves: up to 300mg (Plant DNA 1 protocol) Rice leaves: up to 100mg (Plant DNA 2 protocol) *Difference between Plant DNA 1 and Plant DNA 2 Plant DNA 2 protocol also does the process to remove polysaccharide which is rich in some plant materials. Yield Arabidopsis leaves: 20µg/300mg tissue (Plant DNA 1) Rice leaves: 10µg/100mg tissue (Plant DNA 2) March 16th, 2010 KURABO INDUSTRIES LTD. 21
22 Example 3. (continued) Genomic DNA isolation from plant material PCR analysis Template: 100ng of rice leaf gdna isolated with Plant DNA 1 protocol Target gene: RubisCO gene Amplicon size: 670bp Polymerase: AmpliTaq Gold 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: 25µl Electrophoresis: 10µl of 25µl reaction mixture was loaded on a 1.8% agarose gel. M1 C- C M2 M1: λhindiii size marker C-: Negative control C+: Positive control 1-8: Amplicin of gdna sample isolated with PI-80X M2: φx174 Hin II size marker March 16th, 2010 KURABO INDUSTRIES LTD. 22
23 Contact us KURABO INDUSTRIES LTD. Bio-Medical Department Sales and Marketing Web: March 16th, 2010 KURABO INDUSTRIES LTD. 23
24 Thank you. March 16th, 2010 KURABO INDUSTRIES LTD. 24
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