FavorPrep Plant Genomic DNA Extraction Mini Kit User Manual
Contents
1. Wash Add 500ul of W1 Buffer ethanol added into the column Add 750ul of Wash Buffer ethanol added into the column Centrifuge at full speed 13 000 rpm for 30 seconds Discard the flow through and place the FAPG Column back in the Collection Tube Centrifuge at full speed for 3 minutes to dry the column matrix Important Step The residual liquid can affect the quallity of DNA and inhibit subsequent enzymatic reactions Step 5 DNA Elution Transfer dried FAPG Column into a clean 1 5 ml microcentrifuge tube not provided Add 50 200kl of preheated Elution Buffer into the center of the column matrix Stand for 3 minutes until Elution Buffer absorbed by the matrix Centrifuge full speed 13 000 rpm for 2 minutes to elute purified DNA Important Step For effective elution make sure that the elution solution is dispensed on the membrane center and is absorbed completely Standard elution volume is 200pl If less sample to be used reduce the elution volume 50 150ul to increase DNA concentration 5 FAPGK Troubleshooting Problem Possible Reasons Solution Insufficient Lysis Prolong the incubation time in lysis buffer to obtain higher yields of DNA Insufficient disruption For most of species we recommend grinding with Low yield liquid nitrogen Homogenization should be done thoroughly until the plant2. again for 10 minutes at 4000 X g to dry the column martix Standard elution volume is 1 ml If less sample to be used reduce the elution volume 200 500ul to increase DNA Step 5 DNA Elution concentration If higher DNA yield is required repeat the DNA Elution step to increase DNA recovery and the total elution volume is about 2 ml Transfer dried FAPG Maxi into a clean 50 ml centrifuge tube not provided Add 1 ml of preheated Elution Buffer into the center of the column matrix Stand for 5 minutes until Elution Buffer absorbed by the matrix Centrifuge at 4 000 x g for 3 minutes to elute purified DNA 5 FAPGK Troubleshooting Problem Possible Reasons Solution Low yield Insufficient Lysis Prolong the incubation time in lysis buffer to obtain higher yields of DNA Insufficient disruption For most of species we recommend grinding with liquid nitrogen Homogenization should be done thoroughly until the plant material is ground to a fine powder DNA still bound to the membrane The DNA can be either eluted in higher volumes or by repeating the elution step up to three times Elution buffer should be preheated to 60 C prior to elution To ensure correct pH use supplied elution buffer DNA is degraded Sample was contaminated with DNase Preheat elution buffer to 60 C for 5 minutes to eliminate any possible Dnase Centrifugation speed was to
3. genomic DNA from 50 mg young leave More than 10ug of genomic DNA could be quantified with spectrophotometer and checked by agarose gel Sample 100 mg of plant tissue Yield 5 40ug Operation time lt 60 min 1 FAPGK Kit Contents FAPG1 Buffer FAPG2 Buffer FAPG3 Buffer W1 Buffer Wash Buffer Elution Buffer RNase A 50mg ml Filter Column FAPG Column 1 5 ml Elution tube 2ml Collection tube FAPGK 001 50 preps 24ml 8ml 15ml 22ml 10ml 15ml 500ul 50 pcs 50 pcs 50 pcs 100 pcs FAPGK 001 1 100 preps 50ml 15ml 30ml 44ml 20ml 30ml 840ul 100 pcs 100 pcs 100 pcs 200 pcs Add 30 ml 60 mi ethanol to FAPG3 Buffer before first use Add 8 ml 16ml ethanol to W1 Buffer before first use Add 40 mI 80ml ethanol to Wash Buffer before first use Caution The component contains irritant agent During operation always wear a lab coat disposable gloves and protective goggles 1 PGK 2 Procedure Grind plant sample in liquid nitrogen Lysis FAPG1 Buffer FAPG2 Buffer y FAPG3 Buffer E Filtration centrifuge q J DNA Binding centrifuge Pan Wash W1 Buffer Wash Buffer centrifuge q ka Elute Genomic DNA centrifuge pe 3 FAPGK Protocol Step 1 Tissue Dissociation Cut off 50mg up to 100mg of fresh or frozen plant tissue or 5 mg up to 100 mg of dried sample Grind the sample under liquid nitrogen to a fine powder Transf
4. 2 s 2 FAVORGEN BIOTECH CC FavorPrep Plant Genomic DNA Extraction Mini Kit User Manual Cat No FAPGK 001 50 Preps FAPGK 001 1 100 Preps For Research Use Only v 0905 Australian distributors Fisher Biotec Australia free call 1800 066 077 Ha OE email info fisherbiotec com SEE web www fisherbiotec com 2 DENREN FavorPrep Plant Genomic DNA Extraction Maxi Kit User Manual Cat No FAPGK 002 10 Preps FAPGK 002 1 24 Preps For Research Use Only v 0905 Australian distributors Fisher Biotec Australia free call 1800 066 077 isnadata ta email info fisherbiotec com SEGI web www fisherbiotec com Introduction Genomic DNA Maxi Kit provides a fast and simple method to isolate total DNA genomic DNA mitochondrial and chloroplast from plant tissue and cells In the process sample is distrusted by grinding in liquid nitrogen and lysis buffer incubation The Lysate is treated with RNase A to degrade RNA and filtrated by filter column to remove cell debris and salt precipitations In the presence of binding buffer with chaotropic salt the genomic DNA in the lysate binds to glass fiber matrix in the spin column The contaminants are washed with an ethanol contained wash buffer and finally the purified genomic DNA is eluted by low salt elution buffer or water The protocol does not require DNA phenol extraction and alcohol precipitation The entire procedure can be completed in 60 minutes The p
5. X g for 5 minutes Discard the Filter Column and carefully transfer clarified supernatant in Collection Tube to a new 50 ml centrifuge tube not provided Add 1 5 volumes of FAPG3 Buffer isopropanol added to the cleared lysate and mix immediately by vortexing for 10 seconds For example add 7 5 ml FAPG3 Buffer to 5 ml of lysate Place a FAPG Maxi Column in a 50 ml centrifuge tube Apply the mixture including any precipitate from previous step to the FAPG Maxi Column Centrifuge at 4000 X g for 5 minutes Discard the flow through and place the FAPG Maxi Column back in the Collection Tube Step 3 DNA Binding FAPGK 4 Add 4 ml of W1 Buffer into the column Centrifuge at 4000 X g for 3 minutes Discard the flow through and place the FAPG Maxi Column back in the Collection Tube Add 6 ml of Wash Buffer ethanol added into the column Step 4 Centrifuge at 4000 X g for 3 minutes Wash Discard the flow through and place the FAPG Maxi Column back in the Collection Tube Centrifuge at 4000 X g for 10 minutes to dry the column matrix Optional Step Remove residue pigment If a few pigment remain on the column matrix perform this optional step After Wash Buffer add 4 ml of ethanol 96 100 in the FAPG Maxi Golumn Centrifuge at 4000 X g for 5 minutes Discard the flow through and place the FAPG Maxi Golumn back in the Collection Tubes Centrifuge
6. er it into a microcentrifuge tube not provided For some plant sample we can destruct it without liquid nitrogen Step 2 Lysis Add 400 pi FAPG1 Buffer and 8u RNase A 50 mg ml into the sample tube and mix by vortexing Do not mix FAPG1 Buffer and RNase A before use Incubate at 65 C for 10 minutes During incubation invert the tube every 5 minutes At the same time preheat required Elution Buffer 200uI per sample at 65 C Add 130ul FAPG2 Buffer and mix by vortexing Incubate at ice for 5 minutes Place a Filter Column in a 2 ml Collection Tube Apply the mixture from previous step to the Filter Column Centrifuge for 3 minutes at full speed 13 000 rpm Discard the Filter Column and carefully transfer clarified supernatant in Collection Tube to a new microcentrifuge tube not provided Step 3 DNA Binding Add 1 5 volumes of FAPG3 Buffer ethanol added to the cleared lysate and mix immediately by vortexing for 5 seconds For example add 750ul FAPG3 Buffer to 500 1 lysate Place a FAPG Column in a 2 ml Collection Tube Apply 750ul the mixture including any precipitate from previous step to the FAPG Column Centrifuge at full speed 13 000 rpm for 2 minute Discard flow through in Collection Tube and apply remaining mixture to FAPG Column Centrifuge at full speed 13 000 rpm for 2 minute Discard flow through in Collection Tube FAPGK 4 Step 4
7. material is ground to a fine powder DNA still bound to the membrane The DNA can be either eluted in higher volumes or by repeating the elution step up to three times Elution buffer should be preheated to 60 C prior to elution To ensure correct pH use supplied elution buffer Sample was contaminated with DNase Preheat elution buffer to 60 C for 5 minutes to DNA is eliminate any possible Dnase degraded Centrifugation speed was too high Higher velocities may cause shearing of the DNA The centrifugation maximum speed is at 11 000xg Too much tissue was used Too much tissue was used Reduce the amount of sample material or separate it into multiple tubes Column Insufficient centrifugation clogged Centrifuge again and extend centrifugation time Precipitate was formed at DNA Binding Step Reduce the sample material Before loading the column break up the precipitate in ethanol added lysate by pipetting
8. o high Higher velocities may cause shearing of the DNA The centrifugation maximum speed is at 11 000xg Column clogged Too much tissue was used Too much tissue was used Reduce the amount of sample material or separate it into multiple tubes Insufficient centrifugation Centrifuge again and extend centrifugati on time Precipitate was formed at DNA Binding Step Reduce the sample material Introduction Genomic DNA Mini Kit provides a fast and simple method to isolate total DNA genomic DNA mitochondrial and chloroplast from plant tissue and cells In the process sample is distrusted by grinding in liquid nitrogen and lysis buffer incubation The Lysate is treated with RNase A to degrade RNA and filtrated by filter column to remove cell debris and salt precipitations In the presence of binding buffer with chaotropic salt the genomic DNA in the lysate binds to glass fiber matrix in the spin column The contaminants are washed with an ethanol contained wash buffer and finally the purified genomic DNA is eluted by low salt elution buffer or water The protocol does not require DNA phenol extraction and alcohol precipitation The entire procedure can be completed in 60 minutes The purified genomic DNA is ready for PCR real time PCR Southern blotting and RFLP Quality Control The quality of Plant Genomic DNA Mini Kit is tested on a lot to lot basis The Kits are tested by isolation of
9. r system ensures purified DNA with high yields and a good quality Alternatively buffer FAPGX is provided with the kit also The different detergent in this lysis buffer is suitable for some plant sample with a lot of polysaccharides FAPGK 2 Brief procedure Grind plant sample in liquid nitrogen Lysis FAPG1 Buffer FAPG2 Buffer FAPG3 Buffer l Filtration W centrifuge q DNA Binding MM j centrifuge a m centrifuge q _ Wash W1 Buffer Wash Buffer TI l y Elute Genomic DNA 4 3 FAPGK Protocol Cut off up to 1g of fresh or frozen plant tissue or 50 mg Step 1 up to 100 mg of dried sample Tissue Grind the sample under liquid nitrogen to a fine powder Transfer it into a 15 ml centrifuge tube not provided For some plant Dissociation sample we can destruct it without liquid nitrogen Add 4 ml FAPG1 Buffer or FAPGX Buffer and 50ul Rnase A 10 mg ml into the sample tube and mix by vortexing Do not mix FAPG1 buffer and RNase A before use Incubate at 65 C for 20 minutes During incubation invert the tube every 5 minutes At the same time preheat required Elution Buffer 2 ml per sample at 65 C Step 2 Add 1 ml FAPG2 Buffer and mix by vortexing Lysis Incubate at ice for 5 minutes Place a Filter Column in a 50 ml centrifuge tube not provided Apply the mixture from previous step to the Filter Column Centrifuge at 4000
10. urified genomic DNA is ready for PCR real time PCR Southern blotting and RFLP Quality Control The quality of Plant Genomic DNA Maxi Kit is tested on a lot to lot basis The Kits are tested by isolation of genomic DNA from 50 mg young leave More than 10 ug of genomic DNA could be quantified with spectrophotometer and checked by agarose gel Caution ponent contains irritant agent During operation always wear a lab coat disposable gloves and protective goggles Sample up to 1g of plant tissue Yield 50 300ug Operation time about 60 min 1 FAPGK Kit Contents FAPGK 002 FAPGK 002 1 10 preps 24 preps FAPG1 Buffer 45 ml 110 ml FAPGX Buffer 45 ml 110 ml FAPG2 Buffer 13 ml 30 ml FAPG3 Buffer 30 ml 70 ml W1 Buffer 33 ml 88 ml Wash Buffer 20 ml 45 ml Elution Buffer 30 ml 60 ml RNase A 10mg ml 550 ul 1300 ul Filter Column 10 pcs 24 pcs FAPG Maxi Column 10 pcs 24 pcs Add 60 140 ml ethanol 96 100 to FAPG3 Buffer When first open Add 12 32 ml ethanol 96 100 to W1 Buffer When first open Add 80 180 ml ethanol 96 100 to Wash Buffer When first open Protocol Technical Specification Because of different plant species contain a lot of different metabolites like polysaccharides polyphenolics or proteins Therefore we provide two different lysis buffers for the various plant samples The standard protocol uses FAPG1 Buffer for lysis of plant sample For most of common plant species the buffe
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