Manual - Xcelris Genomics
Contents
1. prokaryotic and eukaryotic genome Genome Mapping and SNP discovery Transcriptome discovery and analysis sRNA analysis and discovery XcelSeq Sanger Sequencing Servi Plasmid PCR Sequencing Services r E coli Culture Sequencing Services Primer Walk Sequencing Services Microbial Identification Service Multilocus Sequence Typing Customised Services SNP Genotyping by SNaPshot Assay Microsatellite Genotyping Golden Gate Assays and Arrays Gene Expression on Real Time PCR Gene expression on Agilent Microarray Affymetix Library construction Xcelris Labs Limited e xce rl S Old Premchand Nagar Road Opp Satyagrah Chhavani Bodakdev Ahmedabad 380015 India Tel 91 79 66197777 Fax 91 79 66309341 genomics Website www xcelrisgenomics com An Abellon Company E mail bdgenomics xcelrislabs com2. 000 rpm Nuclease free 1 5 mland 2 0 ml centrifuge tubes Waterbath equilibrated to 65 C Eguilibrate sterile water or Elution Buffer at 65 C Absolute 96 100 ethanol Optional B mercaptoethanol This is the most robust method for isolation of total cellular mitochondrial chloroplast and genomic DNA Yields are usually sufficient for several tracks on a Southern blot for RFLP mapping Drying allows storage of field specimens for prolonged periods of time prior to processing Samples can be dried overnight ina 45 C oven powdered and stored dry at room temperature To prepare dried samples place 50 mg of dried tissue into a 2 0 ml centrifuge tube and grind using a pellet pestle For critical work such as PCR and cloning pestles are best used a single time then soaked in a dilute bleach solution immediately after use until clean Disposable pestles may be autoclaved several times For standard Southern analysis the same pestle can be reused several times to grind multiple tissue samples by rinsing with ethanol and wiping the surface clean between samples A fine powder will ensure optimal DNA extraction and yield Process four to six tubes as a group till Step 2 and start another set 1 To10 50 mg powdered dry tissue add 900ul Buffer P1 in a 2 0 ml centrifuge tube Optional Add 10ul B mercaptoethanol and vortex vigorously to mix Make sure to disperseall clumps Tip Process in sets of four to six tubes grind add Buffer P1 an
3. L z enomics Table of Contents IHAOCMETICNEE ff 02 OVCE M BPA AE 02 SE ne SLEVY ooo Ako evr MOBA LT 02 KE CONTES or ohh O ONO 03 BONE S AE APR ARR re AM cs 03 HANCODNAMINIRIPETO LOO A S 04 PD DECIMENS 7 Pre SE E 04 EireshlezenEpedmdens Se 0 ooon 06 MEA Sean SLCC gt os 5e P 08 mMoublesrootinmg Guder 2m EE A 09 PMU nd W tranty Se S ene ee ee nen 10 01 Introduction The Plant gDNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from fresh and dried plant tissue samples Up to 100 mg of wet tissue or 30 mg dry tissue can be processed in less than 1 hour The system combines the reversible nucleic acid binding properties of the matrix with the speed and versatility of spin column technology to eliminate polysaccharides phenolic compounds and enzyme inhibitors from plant tissue lysates Purified DNA is suitable for PCR restriction digestion and hybridization technigues There are no organic extractions thus reducing plastic waste and hands on time to allow multiple samples to be processed in parallel Overview If using the Plant gDNA Kit for the first time please read this booklet to become familiar with the procedures Samples are homogenized and lysed in a high salt buffer The DNA is bound to the column while proteins and other impurities are removed by wash buffer The purified DNA is suitable for downstream applications such as endonuclease digestion therm
4. al cycle amplification and hybridization technigues Storage and Stability All components of the Plant gDNA Kit are stable for at least 12 months when stored at 22 C 25 C RNase A should be stored at 4 C During shipment or storage in cool ambient conditions precipitates may form in Buffer P1 and Buffer P2 It is possible to dissolve such deposits by warming the solution at 55 C though we have found that they do not interfere with overall performance 02 xcelris Abellon Gen Plant gDNA Mini Kit Kit Contents Product XG2611 00 XG2611 01 DNA Mini Columns 2 ml Collection Tubes O Z AM 1 1 User Manual Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps Important Dilute Wash Buffer Concentrate with ethanol as follows and store at room temperature Add 8ml XG2611 00 or 60ml XG2611 01 absolute 96 100 ethanol to each bottle Choose the most appropriate protocol to follow Procedures are described for each of dried and fresh or frozen specimens A Dry Specimens For processing lt 50 mg powdered tissue Page 4 DNA yields range from 10 ug to 50 ug per 100 mg drytissue B Fresh Frozen For processing lt 100 mg fresh or frozen tissue Specimens Page 7 DNA yieldis similar to A 03 Plant gDNA Mini Kit Protocol A Dry Specimens Materials suppliedbyuser Microcentrifuge capable ofatleast 13
5. and vortex vigorously Make sure to disperse allclumps DNA cannot be effectively extracted from clumped tissue Tip Process in sets of four to six tubes fill all tubes with liquid nitrogen grind and add Buffer P1 and B mercaptoethanol proceed to Step 2 before starting another set As a starting point use 100 mg tissue per tube and if yield and purity are satisfactory increase to 200 mg 2 Incubate at 65 C for 10 min Mix sample twice during incubation by inverting tube 06 07 Optional If necessary add 5ul of RNase A into the lysate before incubation to remove the RNA Add 140ul Buffer P2 and mix well by vortexing for 10s Centrifuge at 13 000 rpm for 10 min Carefully transfer the supernatant to a clean 2 0 ml tube Add 0 5 volume of Buffer P3 and 0 75 volume 100 ethanol For example 600 ul supernatant 300 ul P3 450 ulethanol and mix well by vortexing for 10s Add 400ul of Buffer BL into the spin column Provided incubate at room temperature for 2 min centrifuge at 12 000 rpm for 2 min and discard the flow through The column is ready and work well for binding DNA Apply the entire sample including any precipitate that may have formed toa DNA column placed in a 2 0ml collection tube supplied Centrifuge the column at 13 000 rpm for 1 min Discard both the 2 0 ml collection tube and the flow through liquid Transfer column to a new collection tube and add 650ul DNA Wash Buffer Centrifuge at 13 000 rpm for 1
6. brate sterile water or Elution Buffer at 65 C e Absolute 96 100 ethanol e Liquid nitrogen for freezing disrupting samples Optional B mercaptoethanol Note Use extreme caution when handling liquid nitrogen This protocol is suitable for most fresh or frozen tissue samples allowing more efficient recovery of DNA However due to the tremendous variation in water and polysaccharide content of plants sample size should be limited to 200 mg Best results are obtained with young leaves or needles The method isolates sufficient DNA for severaltracks on a standard Southern assay To prepare samples collect tissue in a 1 5ml or 2ml centrifuge tube and freeze by dipping in liguid nitrogen with a pair of tweezers to fill the tube Grind the tissue using disposable knots pellet pestles Alternatively one can allow liguid nitrogen to evaporate and then store samples at 70 C for later use For critical work such as PCR and cloning pestles are best used a single time then soaked in a dilute bleach solution immediately after use until cleaning Disposable pestles may be autoclaved several times For standard Southern analysis the same pestle can be reused several times to grind multiple tissue samples by rinsing with ethanol and carefully wiping the surfaces clean between samples 1 Collect ground plant tissue as described start with 100 mg in a 2 0 ml centrifuge tubeandimmediately add 900yl Buffer P1 Optional Add 10ul B mercaptoethanol
7. d B mercaptoethanol and proceed to Step 2 before starting another set Initially do not exceed 50 mg dried tissue Amountcan be increased according to results 2 Incubate at 65 C for 15 min Mixsample twice during incubation by inverting tube Optional If necessary add 5ul of RNase A into the lysate and incubate at room temperature for 5 min before incubation at 65 C to remove the RNA 04 05 Add 140yl Buffer P2 and mix well by vortexing for 10s Centrifuge at 13 000 rpm for 10 min Carefully transfer the supernatant to a clean 2 0 ml tube Add 0 5 volume of Buffer P3 and 0 75 volume 100 ethanol For example 600ul supernatant 300ul P3 450ulethanol and mix well by vortexing for 10s Add 400ul of Buffer BL into the spin column Provided incubate at room temperature for 2 min centrifuge at 12 000 rpm for 2 min and discard the flow through The column is ready and work well for binding DNA Apply the entire sample including any precipitate that may have formed to a DNA column placed in a 2 0 ml collection tube supplied Centrifuge the column at 13 000 rpm for 1 min Discard both the 2 0 ml collection tube and the flow through liguid Transfer column to a new collection tube and add 650ul DNA Wash Buffer Centrifuge at 13 000 rpm for 1 min and discard the flow through liguid Reuse the collection tube in next step below Note Wash Buffer Concentrate must be diluted with absolute 96 100 ethanol priorto use Follow
8. dder 08 Troubleshooting Guide Problems Possible reason Clogged column Carry over of debris Sample too viscous Low DNA Incomplete disruption of yield starting material Poor lysis of sample DNA remains bound to column DNA washed off Problems in downstream applications Salt carry over Ethanol carry over Suggestions Make sure no particulate material is transferred Do not exceed suggested amount of starting material Alternatively increase amounts of Buffers P1 and P2 and use two or more columns per sample For both dry and fresh samples obtain a fine homogeneous powder before adding Buffer P1 Decrease amount of starting material or increase amount of Buffers P1 P2andP3 Increase elution volume to 200 ul and incubate on column at 65 C for 5 min before centrifugation Dilute Wash Buffer by adding appropriate volume of absolute ethanol prior to use eke sed DNA Wash Buffer must be at room temperature Following the second wash spin ensure that the column is dried by centrifuging 5 min at maximum speed Limited Use and Warranty This productis intended for in vitro research use only Not for use in human This product is warranted to perform as described in its labeling and in XcelGen s literature when used in accordance with instructions No other warranties of any kind express or implied including without limitation implied warranties of merchantability or fitness f
9. directions on label Repeat wash step with another 650yul DNA Wash Buffer Centrifuge at 13 000 rpm for 1 min Discard flow through and collection tube use another 2 0 ml collection tubeinnextstep Place the column with the lid open into a new collection tube and centrifuge at 13 000 rpm for 5 min This step is critical for removing residual ethanol that may otherwise be eluted with DNA and interfere with downstream applications Transfer column to a clean 1 5ml microfuge tube Add 100ul Elution Buffer or sterile deionized water pre warmed to 65 C and incubate at room temperature for 3 to 5 min centrifuge at 13 000 rpm for 1 min to elute DNA Smaller volumes will significantly increase DNA concentration but give lower yields Use of more than 200ul of buffer for elution is not recommended Repeat Step 10 with another 100yl Elution Buffer This may be performed using another 1 5 ml microfuge tube to maintain a higher DNA concentration in the first eluate Tip To increase DNA concentration add elution buffer and incubate the column at 60 C 70 C for 5 min before elution Total DNA yields vary depending on type and quantity of sample Typically 10 50 ug DNA with an A A ratio of 1 7 1 9 can be isolated using 50 mg dried tissue B Fresh Frozen Specimens Materials supplied by user Microcentrifuge capable of 13 000 rpm Nuclease free 1 5 mland 2 0 ml centrifuge tubes e Water bath equilibrated to 65 C e Eguili
10. lease read through previous section of this book before using this protocol tle Prepare wet or dry samples by following the standard Protocol in previous sections until loading DNA P3 Ethanol mixture to DNA column Add 400ul of Buffer BL into the spin column Provided incubate at room temperature for 2min centrifuge at 12 000 rpm for 2min and discard the flow through The columnis ready and work well for binding DNA Prepare the vacuum manifold according to manufacturer s instruction and connect the spin column to the manifold Load the DNA P3 Ethanol solution to the column Switch on vacuum source to draw the sample through the column and turn off the vacuum Wash the column by adding 650ul DNA Wash Buffer draw the wash buffer through the column by turn on the vacuum source Repeat this step with another 650pul DNA Wash Buffer Assemble the column into a 2ml collection tube and transfer the column to a micro centrifuge Spin 1 min to dry the column Place the column in a clean 1 5ml microfuge tube and add 100yl Elution Buffer or deionized water Centrifuge at maximum speed for 1 min to elute DNA VASA 5 6 Fig Agarose gel analysis of Plant gDNA purified with XcelGen Plant gDNA mini Kit Lane 1 gDNA Isolated from Rice Lane 2 gDNA Isolated from Maize Lane 3 gDNA Isolated from Mango Lane 4 gDNA Isolated from Fenugreek Lane 5 gDNA Isolated from Pigeon pea Lane 6 gDNA Isolated from Prosopis Lane L Hind III DNA la
11. min and discard the flow through liquid Reuse the collection tube in next step below Note Wash Buffer Concentrate must be diluted with absolute 96 100 ethanol prior touse Follow directions on label Repeat wash step with another 650ul DNA Wash Buffer Centrifuge at 13 000 rpm for 1 min Discard flow through and collection tube and use another 2 0ml collection tube in next step Place the column with the lid open into a new collection tube and centrifuge at 13 000 rpm for 5min This step is critical for removing residual ethanol that may otherwise be eluted with DNA andinterfere with downstream applications Transfer column to a clean 1 5ml microfuge tube Add 100pl Elution Buffer or sterile deionized water pre warmed to 65 C and immediately centrifuge at 13 000 rpm for 1 min to elute DNA Smaller volumes will significantly increase DNA concentration but give lower yields Use of more than 200ul of buffer for elution is notrecommended Repeat Step 10 with another 100yl of Elution Buffer This may be performed using another 1 5 ml microfuge tube to maintain a higher DNA concentration in the first eluate Tip To increase DNA concentration add buffer and incubate the column at 60 C 70 C for 5 min before elution Total DNA yields vary depending on type and quantity of sample Typically 10 50 poDNAwithanA A ratio of 1 7 1 9can beisolated using 100 mg fresh tissue Vacuum Spin Protocol for Plant g DNA Mini Kit Note P
12. or a particular purpose are provided by XcelGen XcelGen s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of XcelGen to replace the products XcelGen shall have no liability for any direct indirect conseguential or incidental damage arising out of the use the results of use or the inability to use it product For technical support or for more product information please visit our website at www xcelrisgenomics com 10 XcelGen Quality Kits made by Xperts Plasmid DNA Isolation Kits Genomic DNA Extraction Kits RNA Extraction Kits Polymerase DNA Ladders DNA Markers Premix Tag dNTP s RAPD kits Agarose Glycerol Tms NA Stabilizers 8 RNA Protectant solutions PrimeX Oligo Synthesis amp Purification Services 10 nmole 25 nmole 50 nmole 100 nmole 200 nmole 1000 nmole NGS Services Denovo Genome Sequencing Whole Genome Resequencing GBS RAD Sequencing Exome Sequencing Amplicon Sequencing Whole Transcriptome Analysis RNA Sequencing Small RNA Sequencing Metagenomics Metatranscriptomics ChIP Sequencing Mitochondrial Sequencing Next Generation Genomic Services on Illumina MiSeq Genotyping by Sequencing Tilling Ecotilling using NGS Genome Database development Services NGS Bioinformatics e In silico Primer Design Microarray Analysis Metagenomics Physical Genetic and QTL mapping Assembly and annotation of
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