FavorPrep Plant Genomic DNA Extraction Maxi Kit User Manual
Contents
1. 3 FAPGK Protocol Cut off 0 5 g 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 Dissociation it into a 15 ml centrifuge tube not provided For some plant sample we can destruct it without liquid nitrogen Add 4 ml FAPG1 Buffer or FAPGX Buffer and 50pl 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 Incubate at ice for 5 minutes Lysis 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 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 Step 3 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 minutes2. Discard the flow through and place the FAPG Maxi Column back in the Collection Tube 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 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 ab
3. FavorPrep Plant Genomic DNA Extraction Maxi Kit User Manual Cat No FAPGK 002 10 Preps FAPGK 002 1 24 Preps For Research Use Only 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 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 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 ig of plant tissue Yiel
4. d 50 300ug Operation time lt 80 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 mi 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 10 pcs FAPG Maxi Column 10 pcs 10 pcs Add 60 140 ml isopropanol 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 Techniical 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 buffer 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 l Lysis FAPG1 Buffer FAPG2 Buffer FAPG3 Buffer Filtration centrifuge z DNA Binding centrifuge C Wash W1 Buffer ay Wash Buffer centrifuge C Elute Genomic DNA
5. sorbed 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 too 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 FAPGK 6
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