EasyPrepTM Plant Genomic DNA Miniprep Manual
Contents
1. Kit Contents Catalog GD02 01 GD02 02 DNA Columns 50 250 Buffer PL1 40 mL 180 mL Buffer CP1 30 mL 130 mL Buffer PL2 12 mL 50 mL Buffer PL3 15 mL 200 mL RNase A 100 uL 500 uL DNA Wash Buffer 5x 20 mL 54 mL Elution Buffer 15 mL 60 mL User Manual 1 1 4 EasyPrep Genomic DNA Miniprep A Fresh Frozen Specimens Before Start e Water bath equilibrated to 65 C e Equilibrate sterile ddH2O or Elution Buffer at 65 C e 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 To prepare samples collect tissue in a 1 5 mL or 2 mL microcentrifuge tube and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube Grind the tissue using disposable kontes pellet pestles which are available from VWR Cat KT749521 0500 Alternatively one can allow liquid 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 multiple2. C for 5 min before centrifugation DNA washed off Dilute Wash Buffer Concentrate by adding appropriate volume of absolute ethanol prior to use page 4 Problems in downstream applications Salt carry over DNA Wash Buffer must be at room temperature Ethanol carry over Following the second wash spin ensure that the column is dried by centrifuging 2 min at maximum speed EasyPrep Genomic DNA Miniprep 11 Bioland Scientific LLC 14925 Paramount Blvd Suite C Paramount CA 90723 USA Tel 877 603 8882 Fax 562 733 6008 Email service bioland sci com order bioland sci com Visit our web at www bioland sci com and learn more about Bioland products
3. impurities are removed by wash buffer The purified DNA is suitable for downstream applications such as endonuclease digestion thermal cycle amplification and hybridization techniques Storage and stability All components of the EasyPrep Plant Genomic DNA Kit are stable for at least 24 months from date of purchase when stored at 22 25 C During shipment or storage in cool ambient conditions precipitates may form in Buffer PL1 and Buffer PL2 It is possible to dissolve such deposits by warming the solution at 37 C though we have found that they do not interfere with overall performance Before Starting Prepare all components and get all necessary materials ready by examining this instruction booklet and become familiar with each steps EasyPrep Genomic DNA Miniprep 3 Important 1 Dilute DNA Wash Buffer with absolute ethanol as follows and store at room temperature e GDO02 01 Add 80 mL absolute ethanol 96 100 e GD02 02 Add 216 mL absolute ethanol 96 100 2 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 5 DNA yields range from 50 pg to 100 ug per 100 mg dry tissue B Fresh Frozen For processing lt 200 mg fresh or frozen Specimens Page 8 tissue DNA yields range from 50 ug to 100 ug per 100 mg dry tissue
4. Table of Contents EasyPrep Plant Genomic DNA NPAC NOM encerrona aaie 3 Miniprep Manual Kit Cs OMMG NMS coiesriceesncocouetecduesteawsdanas7eaneeinawadloseperss 4 Protocols Catalog GD02 01 GD02 02 A Fresh Frozen Specimens 5 B Dry SpecimenSs nnnneneeeeeee re reeeerereeee erenn C Plant rich in polyphenols or polysaccharides 10 Troubleshooting Guide 2 2ccceeeeeeee ene 11 Bioland For research use only March 2013 2 EasyPrep Genomic DNA Miniprep Introduction The EasyPrep Plant genomic DNA 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 techniques 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 EasyPrep Plant Genomic DNA 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
5. l A brief incubation at 65 C may help dissolve the DNA Add 150 wl of Buffer PL3 and 300 pl 96 100 ethanol mix well by vortexing for 5s A precipitation may form but does not interfere with the DNA binding to the column Transfer the sample to the column and centrifuge at 11 000 rpm for 1 min Discard the flow through and put the column back to the collection tube EasyPrep Genomic DNA Miniprep 10 11 12 Add 600 pl DNA Wash Buffer and centrifuge at 11 000 rpm for 20s Discard the flow through and put the column back to the collec tion tube Repeat once Centrifuge the column with the lid open at 11 000 rpm for 1 min This is critical for removing residual ethanol that may interfere with downstream applications Transfer column to a clean 1 5 mL tube Add pre warmed 65 C 100 ul Elution Buffer or sterile ddH20 and immediately centri fuge at 11 000 rpm for 1 min to elute DNA Smaller volumes will sig nificantly increase DNA concentration but give lower yields Use of more than 200 ul of buffer for elution is not recommended Repeat Step 11 with another 100 yl Elution Buffer This may be performed using another 1 5 mL tube to maintain a higher DNA con centration in the first eluate Note To increase DNA concentration add Elution Buffer and incubate the column at 60 70 C for 5 min before elution Total DNA yields vary depending on type and quantity of sample Typi cally 10 50 ug DNA with an Azgo Azgo ra
6. nd vortex vigorously to mix Make sure to disperse all clumps Tip Process in sets of four to six tubes grind add Buffer PL1 and RB mercaptoethanol and proceed to Step 2 before starting another set Initially do not exceed 50 mg dried tissue Amount can be increased according to results 2 Incubate at 65 C for 10 min Mix sample twice during incubation by inverting tube Optional If necessary add 2 ul RNase A into the lysate before incubation to remove the RNA 8 EasyPrep Genomic DNA Miniprep 10 11 Add 200 ul Buffer PL2 and mix well by vortexing for 10s and incu bate on ice for 5 min Centrifuge at 11 000 rpm for 10 min Carefully transfer the supernatant to a clean 2 0 mL tube Add 0 7 volume isopropanol Mix well by vortexing for 5s Centrifuge at 12 000 x g for 2 min to precipitate the DNA This step removes poly saccharides and improves DNA binding ability to the spin column Carefully decant the supernatant and discard avoid dislodging the DNA pellet Inverting the tube on absorbance paper towel for 1 min to drain any residual ethanol The pellet doesn t have to be dry Add 300 ul preheated 65 C ddH2O and vortex for 10s to mix the DNA well A brief incubation at 65 C may help dissolve the DNA Add 150 ul of Buffer PL3 and 300 pl 96 100 ethanol mix well by vortexing for 5s A precipitation may form but does not interfere with the DNA binding to the column Transfer the sample to the column and cen
7. tio of 1 7 1 9 can be isolated using 50 mg dried tissue EasyPrep Genomic DNA Miniprep 7 B Dry Specimens Before start e Waterbath equilibrated to 65 C e Equilibrate sterile ddH2O or Elution Buffer at 65 C 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 in a 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 microcentrifuge tube and grind using a pel let pestle Disposable Kontes pestles work well and are available from VWR Cat KT749521 0500 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 re used 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 1 To 10 50 mg powdered dry tissue add 600 ul Buffer PL1 in a 2 0 mL microcentrifuge tube Optional Add 10 ul R mercaptoethanol a
8. tissue samples by rinsing with ethanol and carefully wiping the surfaces clean between samples EasyPrep Genomic DNA Miniprep 5 Collect ground plant tissue as described start with 100 mg in a 2 0 mL microcentrifuge tube and immediately add 600 ul Buffer PL1 Optional Add 10 ul R mercaptoethanol and vortex vigorously Make sure to disperse all clumps TIP Process in sets of four to six tubes fill all tubes with liquid nitro gen grind and add Buffer PL1 and 2 mercaptoethanol proceed to Step 2 before starting another set As a starting point use 100 mg tis sue per tube If yield and purity are satisfactory increase to 200 mg Incubate at 65 C for 10 min Mix sample twice during incubation by inverting tube Optional If necessary add 2 ul RNase A into the lysate before incubation to remove the RNA Add 140 ul Buffer PL2 and mix well by vortexing for 10s Centri fuge at 11 000 rpm for 10 min Carefully transfer the supernatant to a clean 2 0 mL tube Add 0 7 volume isopropanol Mix well by vortexing for 5s and centrifuge at 12 000 x g for 2 min to precipitate the DNA This step removes poly saccharides and improves DNA binding ability to the spin column Carefully decant the supernatant and discard avoid dislodging the DNA pellet Invert the tube on absorbance paper towel for 1 min to drain any residual ethanol The pellet doesn t have to be dry Add 300 ul preheated 65 C ddH O and vortex for 10s to mix the DNA wel
9. trifuge at 11 000 rpm for 1 min Discard the flow through and put the column back to the col lection tube Add 600 pL DNA Wash Buffer and centrifuge at 11 000 rpm for 20s Discard the flow through and put the column back to the collection tube Repeat once Centrifuge the column with the lid open at 11 000 rpm for 1 min This step is critical for removing residual ethanol that may otherwise be interfere with downstream applications Transfer column to a clean 1 5 ml tube Add pre warmed 65 C 100 ul Elution Buffer or sterile ddH2O and centrifuge at 11 000 rpm for 1 min to elute DNA Smaller volumes will significantly increase DNA concentration but give lower yields Use of more than 200 uL of buffer for elution is not recommended 12 Repeat with another 100 ul Elution Buffer This may be performed us ing another 1 5 mL tube EasyPrep Genomic DNA Miniprep 9 Note 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 Typi cally 10 50 ug DNA with an Azgo Azgo ratio of 1 7 1 9 can be isolated using 50 mg dried tissue Protocol for DNA isolation from plants rich in polyphenols and or polysaccharides 1 Collect ground plant tissue start with 100 mg in a 2 0 mL mi crofuge tube and immediately add 500 uL Buffer CP1 2 Incubate at 65 C for 15 min Mix sample twice during incubation by inverting
10. tube Optional If necessary add 2 uL of RNase into the ly sate before incubation to remove the RNA 3 Add 800 uLchloroform Isoamyl alcohol 24 1 and vortex to mix Cen trifuge at 210 000 x g for 5 min Carefully transfer 300 uL of su perntant to a new tube making sure not to disturb the pellet or any pre cipitates 4 Add 150 uL of Buffer P3 followed by 300 uL of absolute ethanol and vortex to obtain a homogenous mixture A precipitate may form upon addition of ethanol it will not interfere with DNA isolation 5 Apply the entire sample to a DNA column with the collection tube Centrifuge at 13 000 rpm for 1 min to bind DNA Discard the flow through liquid and reuse the collection tube Continue to Step 9 on Page 9 10 EasyPrep Genomic DNA Miniprep Trouble Shooting Guide Possible rea disruption of starting material Problems sons Suggestions Clogged Carry over of Make sure no particulate material debris is transferred column Sample too Do not exceed suggested amount of starting material Alternatively viscous increase amounts of Buffers PL1 and PL2 and use two or more Low DNA Incomplete For both dry and fresh samples yield obtain a fine homogeneous powder before adding Buffer PL1 Poor lysis of sample Decrease amount of starting material or increase amount of Buffers PL1 PL2 and PL3 DNA remains bound to column Increase elution volume to 200 uL and incubate on column at 65
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