FavorPrep. Plant Genomic DNA Extraction Mini Kit. User Manual. For Research Use Only. Cat. No.: FAPGK 001 (50 Preps) FAPGK (100 Preps) v.

Transcription

1 FavrPrep TM Plant Genmic DNA Extractin Mini Kit User Manual Cat. N.: FAPGK 001 (50 Preps) FAPGK (100 Preps) Fr Research Use Only v.0905

2 Intrductin Genmic DNA Mini Kit prvides a fast and simple methd t islate ttal DNA (genmic DNA, mitchndrial and chlrplast) frm plant tissue and cells. In the prcess, sample is distrusted by grinding in liquid nitrgen and lysis buffer incubatin. The Lysate is treated with RNase A t degrade RNA and filtrated by filter clumn t remve cell debris and salt precipitatins. In the presence f binding buffer with chatrpic salt, the genmic DNA in the lysate binds t glass fiber matrix in the spin clumn. The cntaminants are washed with an ethanl cntained wash buffer and finally, the purified genmic DNA is eluted by lw salt elutin buffer r water. The prtcl des nt require DNA phenl extractin and alchl precipitatin. The entire prcedure can be cmpleted in 60 minutes. The purified genmic DNA is ready fr PCR, real-time PCR, Suthern bltting and RFLP. Quality Cntrl The quality f Plant Genmic DNA Mini Kit is tested n a lt-t-lt basis. The Kits are tested by islatin f genmic DNA frm 50 mg yung leave. Mre than 10µg f genmic DNA culd be quantified with spectrphtmeter and checked by agarse gel. Sample: 100 mg f plant tissue Yield: 5~40µg Operatin time: <60 min 1-FAP G K

3 Kit Cntents FAPGK 001 (50 preps) FAPGK (100 preps) FAPG1 Buffer FAPG2 Buffer FAPG3 Buffer* W1 Buffer** Wash Buffer*** Elutin Buffer 24ml 8ml 15ml 22ml 10ml 15ml 50ml 15ml 30ml 44ml 20ml 30ml RNase A (50mg/ml) 500µl 840µl Filter Clumn FAPG Clumn 1.5 ml Elutin tube 2ml Cllectin tube 50 pcs 50 pcs 50 pcs 200 pcs *Add 30 ml/60 ml ethanl t FAPG3 Buffer befre first use. **Add 8 ml/16ml ethanl t W1 Buffer befre first use. ***Add 40 ml/80ml ethanl t Wash Buffer befre first use. Cautin The cmpnent cntains irritant agent. During peratin, always wear a lab cat, dispsable glves, and prtective gggles. FAP G K-2

4 Prcedure 3-FAP G K

5 Prtcl Step 1 Tissue Dissciatin Step 2 Lysis Step 3 DNA Binding Cut ff 50mg (up t 100mg) f fresh r frzen plant tissue r 5 mg (up t 100 mg) f dried sample. Grind the sample under liquid nitrgen t a fine pwder. Transfer it int a micrcentrifuge tube (nt prvided). Fr sme plant sample, we can destruct it withut liquid nitrgen. Add 400 µl FAPG1 Buffer and 8µl RNase A (50 mg/ml) int the sample tube and mix by vrtexing. D nt mix FAPG1 Buffer and RNase A befre use. Incubate at 65 C fr 10 minutes. During incubatin, invert the tube every 5 minutes. At the same time, preheat required Elutin Buffer (200µl per sample) at 65 C. Add 130µl FAPG2 Buffer and mix by vrtexing. Incubate at ice fr 5 minutes. Place a Filter Clumn in a 2 ml Cllectin Tube. Apply the mixture frm previus step t the Filter Clumn. Centrifuge fr 3 minutes at full speed (13,000 rpm). Discard the Filter Clumn and carefully transfer clarified supernatant in Cllectin Tube t a new micrcentrifuge tube (nt prvided). Add 1.5 vlumes f FAPG3 Buffer (ethanl added) t the cleared lysate and mix immediately by vrtexing fr 5 secnds. Fr example, add 750µl FAPG3 Buffer t 500µl lysate. Place a FAPG Clumn in a 2 ml Cllectin Tube. Apply 750µl the mixture (including any precipitate) frm previus step t the FAPG Clumn. Centrifuge at full speed (13,000 rpm) fr 2 minute. Discard flw-thrugh in Cllectin Tube and apply remaining mixture t FAPG Clumn. Centrifuge at full speed (13,000 rpm) fr 2 minute. Discard flw-thrugh in Cllectin Tube. FAP G K-4

6 Step 4 Wash Add 500µl f W1 Buffer (ethanl added) int the clumn. Add 750µl f Wash Buffer (ethanl added) int the clumn. Centrifuge at full speed (13,000 rpm) fr 30 secnds. Discard the flw-thrugh and place the FAPG Clumn back in the Cllectin Tube. Centrifuge at full speed fr 3 minutes t dry the clumn matrix. ---Imprtant Step! The residual liquid can affect the quallity f DNA and inhibit subsequent enzymatic reactins. Step 5 DNA Elutin Transfer dried FAPG Clumn int a clean 1.5 ml micrcentrifuge tube (nt prvided). Add µl f preheated Elutin Buffer int the center f the clumn matrix. Stand fr 3 minutes until Elutin Buffer absrbed by the matrix. Centrifuge full speed (13,000 rpm) fr 2 minutes t elute purified DNA. ---Imprtant Step! Fr effective elutin, make sure that the elutin slutin is dispensed n the membrane center and is absrbed cmpletely. ---Standard elutin vlume is 200µl. If less sample t be used, reduce the elutin vlume (50-150µl) t increase DNA cncentratin. 5-FAP G K

7 Trubleshting Prblem Pssible Reasns/ Slutin Insufficient Lysis Prlng the incubatin time in lysis buffer t btain higher yields f DNA. Lw yield Insufficient disruptin Fr mst f species we recmmend grinding with liquid nitrgen. Hmgenizatin shuld be dne thrughly until the plant material is grund t a fine pwder. DNA still bund t the membrane The DNA can be either eluted in higher vlumes r by repeating the elutin step up t three times. Elutin buffer shuld be preheated t 60 C prir t elutin. T ensure crrect ph, use supplied elutin buffer. DNA is degraded Sample was cntaminated with DNase Preheat elutin buffer t 60 C fr 5 minutes t eliminate any pssible Dnase Centrifugatin speed was t high Higher velcities may cause shearing f the DNA. The centrifugatin maximum speed is at 11,000xg. Clumn clgged T much tissue was used. T much tissue was used. Reduce the amunt f sample material r separate it int multiple tubes. Insufficient centrifugatin Centrifuge again and extend centrifugatin time. Precipitate was frmed at DNA Binding Step Reduce the sample material. Befre lading the clumn, break up the precipitate in ethanl-added lysate by pipetting. FAP G K-6