Kit Manual
Contents
1. Add 60 mL absolute 96 100 ethanol to each bottle GD2413 02 Add 96 mL absolute 96 100 ethanol to each bottle Prepare proteinase K stock solution as following GD2413 00 Add 110 uL Elution Buffer to the vial GD2413 01 Add 1 3 mL Elution Buffer to the vial GD2413 02 Add 1 3 mL Elution Buffer to each vial Biomiga EZgene Insect gDNA Minipre Kit Page 3 Insect gDNA Isolation Protocol Materials to be provided by user Microcentrifuge capable of at least 14 000 x g Nuclease free 1 5 mL or 2 mL microfuge tubes Water bath equilibrated to 65 C Equilibrate sterile ddH O or 10 mM Tris pH 8 5 at 65 C Absolute 96 100 ethanol Chloroform and isoamy alcohol Insect samples preserved in formalin should be rinsed in xylene and then ethanol before processing Note that results obtained with formalin fixed tissues generally depend on age and size of specimen Purified material is usually adequate for PCR amplification but fresh or frozen samples should be used for southern analysis Insects 1 Pulverize no more than 50 mg of tissue in liquid nitrogen with mortar and pestle and place the powder in a clean 1 5 mL microcentrifuge tube If ceramic mortar and pestle are not available homogenize the sample in the microfuge tube using a disposable microtube pestle Proceed to Step 2 below Arthropods and other soft tissue invertebrates 1 Grind no more than 30 mg tissue in liquid nitrog2. C for 5 min before elution Biomiga EZgene Insect gDNA Minipre Kit Page 7 Determination of DNA Quality and Quantity Dilute a portion of the eluted material approximately 10 20 fold in DNA Elution Buffer or 10 mM Tris pH 8 0 Measure absorbance at 280 nm and at 260 nm to determine the Ar o Azgo ratio Values of 1 7 1 9 generally indicate 85 90 purity The concentration of DNA eluted can be determined as follows Concentration 50 pg mL x Absorbance x Dilution Factor Page 8 Biomiga EZgene Insect gDNA Minipre Kit Trouble Shooting Guide Possible Cause Incomplete lysis Suggestions Increase incubation time with Buffer ITL Proteinase K An overnight incubation may be necessary Sample too large Incomplete homogenization Do not use greater than recommended amount of starting material For larger samples divide into multiple tubes Pulverize material as indicated in liquid nitrogen to obtain a fine powder Clogged column See above Poor elution Repeat elution or increase elution volume Incubate the column at 70 C for 5 min before spin Poor binding to column Improper washing Follow protocol closely when adjusting binding conditions DNA Wash Buffer Concentrate must be diluted with ethanol before use Low 260A A280 ratio Extended centrifugation during elution step Poor cell lysis Resin from the column may be present in eluate Avoid centrifugation a
3. Contents Introduction ier tele lets et ei ANA ek 2 Storage and Stability s0ee0ooeeoeeneoo anune anane n naen anna anana anane ane 2 Binding Capaci yore sag ia pa ga a nag a reins a a LA a a a a a aa 2 Kit COntents sa ece NGNE AG NAN GAN GTA hela se KA A aes bins ates 3 Before Starting sos aka Ng Gg E a Aa thle ange e 3 Insect gDNA Isolation Protocol 000es0ee0eseneeneennne nne nenen nne 4 Determination of DNA Quality and Quantity ee eeeeeeeeereeeee 8 Trouble Shooting Guide eee Ga A a Ga NA PEN i e PAGA EA eh 9 Related pi0duc S sarsa saa A en GE a a TA ANAA A E BA Aah neta 10 Limited Use and Warranty 0aseeoeooeonnoeneon naen anana anna nana na nenen 11 Biomiga EZgene Insect gDNA Minipre Kit Page 1 Introduction The EZgene Insect gDNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from insects arthropods and some plant tissue samples rich in polysaccharides The method is suitable for samples frozen or preserved in alcohol or DNE solution and good results can be obtained with formalin preserved material Samples are homogenized and lysed in a high salt buffer and extracted with chloroform to remove polysaccharides Following a rapid alcohol precipitation step binding conditions are adjusted and DNA further purified using ezBind DNA spin columns In this way salts proteins and other contaminants are removed to yield high quality genomic DNA suitable for down
4. en with mortar and pestle and place the powder in a clean 1 5 mL microcentrifuge tube If ceramic mortar and pestle are not available homogenize the sample in the microfuge tube using a disposable microtube pestle Cat SSI 1015 39 amp SSI 1014 39 Addition of a pinch of white quartz sand 50 to 70 mesh Sigma Chemical Co Cat No 9887 will help Proceed to Step 2 below Amount of starting material depends on sample and can be increased if acceptable results are obtained with the suggested 30 mg tissue For easy to Page 4 Biomiga EZgene Insect gDNA Minipre Kit process specimens the procedure may be scaled up and the volumes of all buffers used increased in proportion In any event use no more than 50 mg tissue per ezBind column as DNA binding capacity 100 ug may be exceeded Meanwhile difficult tissues may require starting with less than 30 mg tissue and doubling all volumes to ensure adequate lysis Add 350 uL Buffer ITL followed by 25 uL Proteinase K 25 mg mL Vortex briefly to mix and incubate at 60 C for a minimum of 30 min or until entire sample is solubilized Actual incubation times vary and depend on elasticity of tissues Most samples require no more than 4 hours Alternatively an overnight incubation at 37 C will produce adequate results To the lysate add 350 uL chloroform isoamyl alcohol 24 1 and vortex to mix Centrifuge at 10 000 x g for 2 min at room temperature Carefully transfer the upper aqueo
5. ollection tube and wash by adding 650 yL DNA Wash Buffer diluted with absolute ethanol Centrifuge 10 000 x g for 30 s Discard flow through liquid and re use collecting tube in next step Note DNA Wash Buffer is provided as a concentrate and must be diluted with absolute ethanol as indicated on the bottle and page 3 If refrigerated the diluted DNA Wash Buffer must be brought to room temperature before use 10 Repeat step 9 with a second 650 pL DNA Wash Buffer diluted with ethanol 1 e Discard liquid and collection tube Insert the column to a new collection tube with the lid open and centrifuge the column at 13 000 x g for 2 min at room temperature Note This step is critical in removing traces of ethanol that will interfere with downstream applications Place column into a clean 1 5 mL microfuge tube not suplied To elute DNA add 50 wL 100 uL of Elution Buffer 10 mM Tris HCL pH 8 5 preheated to Page 6 Biomiga EZgene Insect gDNA Minipre Kit 60 C 70 C directly onto the ezBind matrix Allow soaking for 2 min at room temperature Centrifuge at 13 000 x g for 1 min to Elute DNA 12 Repeat elution step with a second 50 uL 100 uL Elution Buffer Note Typically a total of 5 15 ng DNA with absorbance ratio A260 A280 of 1 7 1 9 can be obtained Yields vary depending on source and quantity of starting material used Note To increase DNA Yield add Elution buffer and incubate the column at 60 C 70
6. stream applications such as endonuclease digestion thermal cycle amplification and hybridization techniques Storage and Stability All components of the EZgeneTM Insect gDNA Kit except the Proteinase K and RNase A should be stored at 22 C 25 C Once reconstituted in water Proteinase K should be stored 20 C Under at these conditions DNA has successfully been purified and used for PCR after 12 months of storage Store RNase A at 4 C All EZgene Insect gDNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at 22 C 25 C Binding Capacity Each ezBind DNA column can bind approximately 100 ug DNA Using greater than 30 mg tissue is not recommended Page 2 Biomiga EZgene Insect gDNA Minipre Kit Kit content Product GD2413 00 GD2413 01 GD2413 02 Preps 4 50 250 ezBind DNA Columns 4 50 250 2 mL Collection tubes 8 100 500 Buffer ITL 2 mL 20 mL 100 mL Buffer BL 2 mL 20 mL 100 mL Buffer KB 2 8 mL 28 mL 135 mL Proteinase K 2 mg 30 mg 5 x 30 mg RNase A 20mg mL 25 uL 270 uL 1 35 mL DNA Wash Buffer 2 mL 15 mL 3 x 24 mL Elution Buffer 1 mL 15 mL 70 mL User Manual 1 1 1 Before Starting Please read the entire booklet to become familiar with the EZgene Insect DNA Kit protocol Dilute DNA Wash Buffer Concentrate with absolute ethanol as follows and store at room temperature GD2413 00 Add 8 mL absolute 96 100 ethanol GD2413 01
7. t gDNA kit 250 420 00 GD2414 02 250 420 00 GD2415 02 250 420 00 n Nn Eat GD2416 01 Fungal gDNA kit 90 00 GD2416 02 Fungal gDNA kit 250 420 00 Page 10 Biomiga EZgene Insect gDNA Minipre Kit Limited Use and Warranty This product is 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 Biomiga 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 for a particular purpose are provided by Biomiga Biomiga s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of Biomiga to replace the products Biomiga shall have no liability for any direct indirect consequential or incidental damage arising out of the use the results of use or the inability to use it product Biomiga EZgene Insect gDNA Minipre Kit Page 11 For technology support or learn more product information please visit our website at www biomiga com or contact us at 858 603 3219 Page 12 Biomiga EZgene Insect gDNA Minipre Kit
8. t speeds higher than specified The material can be removed from the eluate by centrifugation it will not interfere with PCR or restriction digests Increase incubation time with Buffer ITL An overnight incubation may be necessary Trace protein contaminants remain No DNA eluted Poor cell lysis Following step 8 wash column with a mixture of 300 uL Buffer BL 300 uL ethanol before proceeding to step 9 Increase incubation time with Buffer ITL An overnight incubation may be necessary Incomplete homogenization Absolute ethanol not added before adding sample to column Pulverize starting material as indicated in liquid nitrogen to obtain a fine powder Before applying DNA sample to column add Buffer BL and absolute ethanol No ethanol added to DNA Wash Buffer Concentrate Dilute Wash Buffer with the indicated volume of absolute ethanol before first use Biomiga EZgene Insect gDNA Minipre Kit Page 9 Related EZgene Products Catalog Product Name Preps Price GD2211 02 250 420 00 Na BA GD2311 01 Blood gDNA mini kit 90 00 GD2311 02 Blood gDNA mini kit 250 420 00 GD2312 01 Blood gDNA midi kit 10 9000 GD2312 02 Blood gDNA midi kit 25 200 00 v njo GD2314 01 Blood gDNA maxi kit 160 00 GD2314 02 Blood gDNA maxi kit 360 00 GD2411 02 250 495 00 GD2412 02 250 420 00 n An ajola GD2413 01 Insect gDNA kit 90 00 GD2413 02 Insec
9. us phase to a clean 1 5 mL microfuge tube Avoid the milky interface containing contaminants and inhibitors Note This step will remove much of the polysaccharides and proteins from solution and improve spin column performance downstream If there is very few upper aqueous phase present after centrifugation add 200 uL of Buffer and vortex to mix Centrifuge as above and transfer the upper aqueous phase to tube Add 1 volume of Buffer BL followed by 5 pL RNase A vortex at maxi speed for 15 s Incubate at 70 C for 10 min Add 1 volume of absolute ethanol room temperature 96 100 and mix well by vortexing at maxi speed for 15 s Tips 500 uL upper aqueous solution add 500 uL Buffer BL and 500 uL of absolute ethanol Biomiga EZgene Insect gDNA Minipre Kit Page 5 Apply 700 uL of the mixture from step 5 including any precipitation that may have formed to the ezBind DNA column Centrifuge at 10 000 x g for 1 min at room temperature Discard flow through liquid and re use collection tube Place ezBind DNA column back into the same collection tube apply the remaining of mixture into the column and centrifuge as above Discard flow through liquid and collection tube Place the column into another a new 2 mL collection tube supplied and wash by adding 500 uL Buffer KB Centrifuge at 10 000 x g for 30 s Discard flow through liquid and re use collecting tube in next step Place column into the c
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