FavorPrep Plant Genomic DNA Extraction Mini Kit User Manual
Contents
1. Buffer 24ml 50ml FAPG2 Buffer 8ml 15ml FAPG3 Buffer 15ml 30ml W1 Buffer 22ml 44ml Wash Buffer 10ml 20ml Elution Buffer 15ml 30ml RNase A 50mg ml 500 ul 840 ul Filter Column 50 pcs 100 pcs FAPG Column 50 pcs 100 pcs 1 5 ml Elution tube 50 pcs 100 pcs 2ml Collection tube 100 pcs 200 pcs Add 30 ml 60 ml ethanol to FAPG3 Buffer before first use Add 8 ml 16ml ethanol to W1 Buffer before first use Add 40 ml 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 FAPGK 2 Procedure Grind plant sample in liguid nitrogen l Lysis FAPG1 Buffer FAPG2 Buffer FAPG3 Buffer Filtration centrifuge DNA Binding centrifuge Wash W1 Buffer s Wash Buffer centrifuge Elute Genomic DNA centrifuge 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 liguid nitrogen to a fine powder Transfer it into a microcentrifuge tube not provided For some plant sample we can destruct it without liguid nitrogen Step 2 Lysis Add 400ul FAPG1 Buffer and 4 pI RNase A 100 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 tube2. FavorPrep Plant Genomic DNA Extraction Mini Kit User Manual Cat No FAPGK 001 50 Preps FAPGK 001 1 100 Preps For Research Use Only 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 liguid 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 yg of genomic DNA could be quantified with spectrophotometer and checked by agarose gel Sample 100 mg of plant tissue Yield 5 30g Operation time lt 60 min 1 FAPGK Kit Contents FAPGK 001 FAPGK 001 1 50 preps 100 preps FAPG1
3. every 5 minutes At the same time preheat required Elution Buffer 200 ul 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 lysate Place a FAPG Column in a 2 ml Collection Tube Apply 700ul 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 Wash Add 500u 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 Discard the flow through and place the FAPG Column back in the Collection Tube Centr
4. ifuge at full speed for 3 minutes to dry the column matrix Step 5 DNA Elution Standard elution volume is 100ul If less sample to be used reduce the elution volume 30 50ul to increase DNA concentration If higher DNA yield is reguired repeat the DNA Elution step to increase DNA recovery and the total elution volume is about 200ul Transfer dried FAPG Column into a clean 1 5 ml microcentrifuge tube not provided Add 100u of preheated Elution Buffer into the center of the column matrix Stand for 3 5 minutes until Elution Buffer absorbed by the matrix Centrifuge full speed 13 000 rpm for 30 seconds to elute purified DNA 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 liguid 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 Sample was contaminated with DNase Preheat elution buffer to 60 C for 5 minutes to DNA is eliminate any possible Dnase deg
5. raded 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 FAPGK 6
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