Table of Contents. I. Description II. Kit components III. Reagents and instruments IV Storage V. Protocol...

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1 Cat.# 9084 v Table of Contents I. Description... 2 II. Kit components... 2 III. Reagents and instruments... 2 IV Storage... 2 V. Protocol...3 VI. Schematic Representation of Standard Protocol...4 Vll. Q & A...5 DNA Isolation Kit Cat. Dr. GenTLE (from whole blood)... #9081 PCR related products TaKaRa Taq... #R001 TaKaRa Ex Taq...#RR001 TaKaRa LA Taq...#RR002 PCR Amplification Kit... #R011 LA PCR Kit Ver #RR013 RNA PCR Kit Ver #R019 RNA LA PCR Kit Ver #RR012 LA PCR in vitro Cloning Kit...#RR015 LA PCR in vitro Mutagenesis Kit...#RR016 Rapid DNA ligation system DNA Ligation Kit Ver. 1 (rapid ligation system)... #6021 DNA Ligation Kit Ver. 2 (rapid ligation system)... #6022 DNA Blunting Kit...#6025 DNA Extraction Cartridges SUPREC -01 (elution from gel slices)...#9040 SUPREC -02 (concentration & purification)...#9041 DNA labeling systems DNA 5' end-labeling kit MEGALABEL...#6070 Ladderman Labeling Kit (Bca DNA polymerase / random-primed)...#6046 Random Primer Labeling Kit (Klenow fragment)...#6045 DNA Sequencing Systems TaKaRa Taq Cycle Sequencing Kit... #R014 TaKaRa Taq Cycle Sequencing Core Kit... #R018 Ladderman Dideoxy Sequencing Kit (for ATP labeling)... # DEAZA Sequencing Kit (Klenow fragment)...#6015 1

2 I. Description: Dr. GenTLE (50 preps)... Cat.#9084 Dr. GenTLE (Gene Trapping by Liquid Extraction) is designed to enable the rapid extraction of highly-purified genomic DNA from yeast. The isolated DNA can be applicable to various subsequent reactions. The protocol takes just around 30 minutes and highly pure intact DNA can be obtained with a simple procedure. The standard protocol describes the reaction using 1.0~1.5 ml of cultures. But this kit can also be used with a larger amount of samples. This kit is composed of three GenTLE Yeast solutions (GenTLE Yeast solution I, II, III) and TE Buffer. The protocol consists of the following 6 steps: 1) pelleting cells by centrifugation 2) resuspension of cells in GenTLE Yeast Solution I followed by addition of GenTLE Yeast Solution II 3) incubation at 70 C for 10 min. followed by addition of GenTLE Yeast Solution III 4) centrifugation followed by precipitation of DNA from the supernatant using isopropanol 5) washing pellet with 70% ethanol 6) resuspension of the DNA As this kit utilizes a unique lysis buffer technology, no need to use lytic enzymes, proteinase K, or phenol. Typical yields are 5-20 µg from a 1.5 ml yeast culture such as Saccharomyces cerevisiae or Schizosaccharomyces pombe. The DNA extracted with this kit can be used directly for restriction enzyme digestion and PCR. II. Kit components: 1.GenTLE Yeast Solution I ml 2.GenTLE Yeast Solution II... 3 ml 3.GenTLE Yeast Solution III ml 4.TE Buffer ml III. Required reagents & instruments other than kit: Centrifuge (applicable to a tube) Isopropanol 70% Ethanol Tube (applicable to an used sample) Micropipette and tips(autoclaved) Heatblock (for heating at 70 C) IV.Storage: 4 C. Note: GenTLE Yeast Solution II may generate precipitate during the storage at 4 C. In case that precipitate forms, warm at 37 C for 1-5 min. to bring the material back into solution. The solution should be used after the precipitate is completely dissolved. 2

3 V. Protocol: 1) Add 1.0~1.5 ml* of yeast culture into a microcentrifuge tube and centrifuge at 12,000 rpm for 30 sec. (*When larger volume of the sample is treated, please see the following Technical Note 4.) 2) Discard the supernatant with a micropipette, taking care to remove as much media as possible. 3) Add 540 µl of GenTLE Yeast Solution I to the pellet and completely resuspend the cells either by vortexing or pipetting up and down repeatedly with a micropipette. 4) Add 60 µl of GenTLE Yeast Solution II and mix thoroughly by vortexing. 5) Heat at 70 C for 10 min. 6) Add 300 µl of GenTLE Yeast Solution III and mix gently by converting the tube. Do not vortex. 7) Place the tube on ice for 2 min. and then mix again gently by converting the tube. Do not vortex. 8) Centrifuge at 12,000 rpm for 5 min. at 4 C. 9) Transfer the supernatant into a fresh microcentrifuge tube. Add 600 µl of isopropanol and mix by vortexing to precipitate the DNA*. *NOTE: This method until Step 9 will not produce an obvious amount of precipitated DNA as seen with standard genomic preparations, but will form a pellet after centrifugation at Step ) Centrifuge at 12,000 rpm for 5 min. at 4 C and then discard the supernatant. White DNA pellet should be visible. 11) Wash the DNA pellet with 200 µl of cold 70% ethanol and centrifuge again at 12,000 rpm for 5 min. at 4 C. 12) Discard the supernatant and dry the DNA pellet either by air or vacuum using a speed-vacuum. 13) Dissolve the pellet in 50 µl of TE Buffer or other solutions appropriate to subsequent reactions. NOTE: The resuspended DNA should be stored at 4 C or -20 C. Technical Notes: 1. The yield of this kit should be approximately 7 µg from 1 ml of yeast culture. Lower yields may be observed if the culture contains less yeast cells or the culture is not fresh. 2. The purity of the isolated yeast genomic DNA with this kit is OD 260/280 = Although this protocol does not depend on the growth phase of the culture, highest yields will be obtained using an overnight culture grown to late log - early stationary phase in a low protein medium. 4. The volume in this protocol can be increased up to 10 ml of overnight culture. When treating volumes more than 1.5 ml, Step 1 should be performed in a 15 ml centrifuge tube. In this case, transfer the sample into a microcentrifuge tube after resuspension in Step For most preps, the RNase treatment included in the kit protocol is sufficient for removal of RNA. For those strains that have an exceptionally high level of RNA, any standard RNase treatment is recommended after the genomic DNA is isolated. 6. Plasmid can be extracted with this kit, but it is intermingled with genomic DNA. 3

4 VI. Schematic Representation of Standard Protocol: Yeast Cultures Centrifuge. Pellets of Yeast Supernatant Resuspend in GenTLE Yeast Solution I. Add GenTLE Yeast Solution II. Incubate at 70 C for 10 min. Add GenTLE Yeast Solution III. Cool on ice for 2 min. Centrifuge. Genomic DNA Precipitate with isopropanol. Wash with 70% ethanol and dry. Subsequent reaction 4

5 VIl. Q & A : Q : Is this kit avairable for bacteria? A : Although this kit is optimized for yeast, it is also applicable to extract the genomic DNA from bacteria. The gram-negative bacteria are comparatively suitable for this kit. The DNA extracted from these bacteria with this kit can be used for PCR and restriction enzyme digestion. However in many cases, both of the yield and the purity of the DNA from these bacteria may be lower than those from yeast. On the other hand, gram-positive bacteria are not very suitable for this kit, because these bacteria contain a large amount of polysaccharides in the cell wall. The yield and the purity of the DNA extracted from these bacteria may not be good. But in most cases, the extracted DNA can be used as a template for PCR. Q : What bacteria strains were confirmed to be availrable with this kit? A : The gram-negative bacteria tested: Escherichia coli, Thermus aquaticus, Pseudomonas aeruginosa, Flavobacterium okeanokoites, Enterobacter aerogenes, Vibrio parahaemolyticus The gram-positive bacteria tested: Micrococcus luteus, Staphylococcus aureus, Arthrobacter luteus, Bacillus amyloliquefaciens, Bacillus subtilis, Lactobacillus sp., Leuconostoc sp. 16S rdna region (1.5 kbp) can be amplified from all the DNA extracted from these bacteria using the universal primers. Related products: For DNA Isolation from whole blood; Dr. GenTLE (from whole blood)... Cat.#9081 Note: For research use only. Not for use in diagnostic or therapeutic procedures. 5