EZ-Genotyping™ Kit - System Biosciences
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1. 6 ver 1 140115 www systembio com EZ Genotyping Kit Cat GE2XXA 1 WT Samples 1 and 3 KO Mono allele Samples 4 5 6 7 8 9 10 KO Bi allele Sample 2 Figure 4 Genotyping results for validation of allele status in gene targeting assay using homologous recombination HR targeting vector 4 Positive controls Positive Controls 1 5 Agarose Gel 0 a amp Vy amp V S Ss gt FST FS Sos PS bo oe 2 Th U Mouse 94 Cx3 min 94 Cx30 s 60 Cx30 s 72 Cx20 s 30 cycles 72 Cx5 min gt Incubate at 4 C Mouse Rat Human 212 bp Figure 5 PCR results for positive control primers in EZ Genotyping Kit demonstrates robust and consistent amplicon formation in different mammalian samples Vil Technical Support Information For more information about SBI products and to download manuals in PDF format please visit our web site http Awww systembio com For additional information or technical assistance please call or email us at System Biosciences SBI 265 North Whisman Rd Mountain View CA 94043 Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual Vill Licensing and Warranty Limited Use License Use of the2. EZ Genotyping Kit i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described
3. SBI System Biosciences EZ Genotyping Kit Cat GE2XXA 1 User Manual Store kit at 20 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual EZ Genotyping Kit Cat GE2XXA 1 Contents l WINtrOdUCHIONY a Aiwe ate ave eee 1 A Overview of EZ Genotyping Kit 1 B Highlights of EZ Genotyping Kit sser 2 Il Product Information ccccccceeeeeeeeeececeeeeeaeeeeeeeseeeeeseeaeeseneeee 2 Table 1 Components of EZ Genotyping Kit GE200A 1 2 II Genotyping Protocol ccccccceeeeeeeeeeeeeeeeeseeeeeseeeetaeeeeeeeees 3 A Overview Of Workflow cccceeeecceeeeeeeeeeeeeeeeseeeeeseaeeteaeeeeaes 3 Figure 1 Workflow of the EZ Genotyping Kit 3 BE PfOtOCOl niad n e e a eet a atas 3 Table 2 Recommended Amounts of CellExtract Solution for Different Well Sizes 3 IV Troubleshooting cceeeceeeeeeeeneeeeeeeeceaeeeseaeeeeaeeseeeeseaaeeneneeee 5 Table 3 Recommended Solutions for Troubleshooting 5 Ve Applications indii eiaa a aa a aa a atai 5 VI Performance Data and Applications cccceceseeeeeeseeeees 5 Figure 2 Comparison of PCR amplification in genomic DNA extracts using EZ Genotyping Kit vs purified genomic DNA 6 Figure 3 Successful amplification and allelic discrimination of multiple genomic markers DNA f
4. UME Forward Primer 10 uM 1 0 uL 1 0 uL 10 uM of Reverse Primer 10 uM 1 0 uL each Template 2 uL 2 uL 6 uL 7 uL Total Volume 20 uL 20 uL PCR Conditions 1 Denature the template at 94C for 3 min 2 Denature at 94C for 30 s Anneal primers at 60C for 30 s Extension at 72C for 10 s per every 100bp 32 34 cycles of amplification are recommended 3 Store at 4C indefinitely The percentage of agarose gel 1 2 5 to use for a given PCR product size Resolution of Linear DNA on Agarose Gels Page 4 ver 1 140115 www systembio com EZ Genotyping Kit Cat GE2XXA 1 IV Troubleshooting Table 3 Recommended Solutions for Troubleshooting PROBLEM REASON POSSIBLE SOLUTIONS A Prepare PCR reaction on ice B Hotstart by pre heating Mispriming the thermocycler to 94 degree before adding samples or use a hotstart specific Taq polymerase Non specific bands Run positive control Design and order new Extracts contain No band no genomic i i o bands DNA primers in case primer degradation or incorrrectness Primers errors Run a dilution series of DNA samples including a non diluted sample Poor Too much or too Amplification little DNA Increase the number of cycles or decrease the annealing temperature Weak or absent Low number of bands cycles V Applications The follow applications have been successfully tested by using the EZ Genotyping Kit 1 Genotype analysis in mixed cl
5. in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a credit This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a credit limited to the actual purchase price SBI s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 2014 System Biosciences SBI All Rights Reserved Page 8 ver 1 140115 www systembio com
6. ingerprinting 6 Figure 4 Genotyping results for validation of allele status in gene targeting assay using homologous recombination HR targeting vector esssessseesseeesseessrresresreeee 7 Figure 5 PCR results for positive control primers in EZ Genotyping Kit demonstrates robust and consistent amplicon formation in different mammalian samples 7 VII Technical Support Information cceesceeeeeeeeeeeeeeseeeees 7 VIII Licensing and Warranty cc cccccccecseeeesseeeeeneeeseneeesnaeeeeeeesees 8 I Introduction A Overview of EZ Genotyping Kit The EZ Genotyping Kit provides reagents to effectively determine the genotypes of target alleles in most mammalian samples using a robust endpoint PCR reaction The complete kit contains sufficient reagents for genomic DNA extraction from 50 samples and subsequent PCR reactions This genotyping kits offers a fast and streamlined solution for genotyping analysis of cultured mammalian cells A major advantage of the kit is that genomic DNA 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual extraction is carried out in a single tube mitigating chances for sample cross contamination for subsequent PCR analysis The kit is amenable to high throughput based screening and suitable to several downstream applications such as identification of genotypes in mixed clonal cell populations DNA fingerprinting and cell identit
7. onal cell populations 2 DNA fingerprinting analysis 3 Culture cell identity and contamination checking VI Performance Data and Applications 1 Reliable and unambiguous genotyping results Reliability and consistency are important factors in choosing a genotyping method Due to less than optimal genotyping conditions for many commercial and homemade protocols the PCR analysis sometimes lead to weak or ambiguous bands SBI s EZ Genotyping kit provides a reliable solution for amplifying various amounts of genomic DNA in extracts compared to those of purified DNA templates Figure 2 below 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Using 2xPCR Mix Extract Volume for PCR Amountof purified gDNA for PCR amp MM o Yo MM SS amp amp gt gt Y g D Y S S SF Experiment Conditions 94 Cx3 min 94 Cx30 s 60 Cx30 s 72 Cx20 s 30 cycles 72 Cx5 min gt Incubate at 4 C Figure 2 Comparison of PCR amplification in genomic DNA extracts using EZ Genotyping Kit vs purified genomic DNA 2 Genes with different allele sizes and chromosome locations can be successful amplified Figure 3 Successful amplification and allelic discrimination of multiple genomic markers DNA fingerprinting 3 Confirmation of mono or bi allelic modifications by genotyping Si 2345 at LERIU 678910 KO 1281 bp WT 1067 bp Page
8. systembio com EZ Genotyping Kit Cat GE2XXA 1 Ill Genotyping Protocol A Overview of Workflow Workflow Sample Preparation Results J 6 i f C Cultured cells Gel Electrophoresis Incubate at 95 C PCR 5 min 2 hours 1 hour Figure 1 Workflow of the EZ Genotyping Kit B Protocol Example of cells in culture using a 24 well plate volume can be scaled up down depending on plate size Remove culture medium and wash cells 2x in each well with 0 5 ml of PBS Remove all of the PBS from the wells Add 100 ul of CellExtract Solution to each well Dislodge the cells in each well by pipetting the solution up and down several times to form a cell extract Transfer the cell extract to an Eppendorf tube and pipette up and down the extracts an additional 3 5 times Heat the extract at 95 C for 2 min Centrifuge the heated extract at 14 000 RPM for 2 min Save the supernatant as it contains the genomic DNA which is ready for PCR amplification OO N o Ole NS Table 2 Recommended Amounts of CellExtract Solution for Different Well Sizes CULTURE SURFACE CELL CELLEXTRACT DISH AREA cm NUMBER SOLUTION nL 96 Well 48 Well 24 Well 12 Well 6 Well 35mm 60mm T25 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Add the following reagents to a 0 2 or 0 5 ml PCR tube in the following order POSITIVE CONTROL 2xPCR Mix 10 uL 10 uL REAGENT VOL
9. y contamination analysis B Highlights of EZ Genotyping Kit 1 Easy to use no need for overnight digestion or column purification of genomic DNA 2 Complete Package contains both genomic DNA template preparation and 2X PCR Master Mix for PCR reaction 3 Robust Results the complete solution was tested to successfully amplify multiple targets on different chromosomes 4 Reliable positive control primers are included to ensure successful preparation and subsequent amplification of genomic targets to assure the absence of false negative results 5 Economical The kit provides enough reagents for carrying out 50 genotyping reactions Il Product Information Product Name EZ Genotyping Kit Catalog Number GE200A 1 Shipping Condition Blue Ice or Dry Ice Storage Condition 20 C Table 1 Components of EZ Genotyping Kit GE200A 1 COMPONENTS DESCRIPTION Simple one step CellExtract Solution solution for sample GE210A 1 DNA extraction without DNA purification 2xPCR Mix contains all the components e g 2x PCR Master Mix dNTPs MgCl2 Taq necessary to perform PCR reaction 10uM of each primer Positive Control designed to give 212 Primer Set bp PCR product in ul each most species Note Please avoid multiple freeze thaw cycles for the 2X PCR Master Mix The 2X PCR Master Mix can be kept at 4 C after thawing for a period of 3 month without significant change in performance Page 2 ver 1 140115 www
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