Random Primer DNA Labeling Kit

Table of contents

v.0706

I II III IV

Description Principles Procedure Effect of the amount of template DNA Measurement of incorporation yields by DE81-filter binding assays
Random Primer DNA Labeling Kit (30 reactions)

Description:

The Random Primer DNA Labeling Kit is designed to yield DNA probes with high specific activity for hybridization, and can be used to label DNA with [ -32P], [ -35S] or [3H] dCTP. This kit is based on a modified method of Feinberg and Vogelstein1) 2), utilizing 9 mer random oligonucleotide primers and the Exo-free Klenow fragment of E.coli DNA polymerase I which lacks 3' 5' exonuclease activity. Each reaction is capable of generating DNA probes of high specific activity of 1x109 dpm/g in as little as 2-3 minutes. It overcomes many of the disadvantages of the conventional nick translation procedure as shown in Table 1. Kit Components:
Random primer (9 mer)...60 l 10x buffer....75 l dNTP mixture (0.2 mM each dGTP, dATP, and dTTP)..75 l Exo-free Klenow fragment (2 units/ul)..30 l Control DNA solution ( DNA -Hin d III digest (25 ng/ l)).10 l

Storage:

Reagents not supplied in the kit: Distilled sterilized water TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM EDTA) Labeled dCTP solution; This kit is designed for use with [ -32P]dCTP (~111 TBq (3,000 Ci) /mmol), however, [ -35S]- and [3H]dCTP can also be used. References: 1) Feinberg,A.P. and Vogelstein,B. (1983) Anal. Biochem. , 132, 6-13. 2) Feinberg,A.P. and Vogelstein,B. (1984) Anal. Biochem. , 137, 266-267. 3) Clark. J. M. , Joyce. C. M. , and Beardsley G. P. ,(1987), J. Mol. Biol. , 198, 123-127.
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TAKARA BIO INC.

I. Principles:
DNA probes with high specific activity are required for the detection of specific DNA sequences in many hybridization experiments. The random primer labeling procedure reported by Feinberg and Vogelstein 1) 2) produces probes with very high specific activity from small amounts of DNA (10-20 ng) and can be used to label DNA fragments even directly in low melting temperature agarose gel slices. As illustrated in Fig. 1, the DNA to be labeled is heat denatured to produce single-stranded template. The random primers anneal to the single-stranded template DNA and are extended by the Klenow fragment as it incorporates labeled and unlabeled nucleotides. Figure 1. The principle of random primer labeling Table 1
Specific activity of product Template impurities Purification after the reaction Amount of template required ~108 dpm/ g Inhibited by the presence of agarose Removal of unused dNTP is required 1 g Incorporation rates Nick translation method Decreases after prolonged incubation DNA probes with high specific activity are obtained in a short time. Unaffected by prolonged incubation ~109 dpm/ g Relatively unaffected by the presence of agarose Removal of dNTP is not required 25 ng Random primer method
These comparisons are based on the results of multiple control experiments using a DNA- Hind III template that was labeled with 50 Ci of [ -32P] dCTP (3000 Ci/mmol)

II. Procedure:

1) Combine the following reagents in a microcentrifuge tube and bring the total volume to 14 l with distilled sterilized water or TE buffer.
Template DNA.. 10 ng - 1 g Random primer... 2 l Sterile distilled water or TE buffer.. x l Total...14 l
2) Heat at 95 for 3 minutes and then cool on ice for 5 minutes. 3) Add 2.5 l of 10 x buffer, 2.5 l of the dNTP mixture, and 5 l of labeled dCTP. *2 (1.85MBq, 50 Ci) 4) Add 1 l of Exo-free Klenow fragment and incubate at 37 for 10 minutes.*3 5) Inactivate the enzyme by heating the mixture at 65 for 5 minutes or adding EDTA in a final concentration of 30 mM. 6) Heat at 95 for 3 minutes and then cool on ice. 7) Use an appropriate volume as hybridization probe. (When necessary, unincorporated dCTP is removed by gel filtration or by ethanol precipitation.) *1 Template DNA having the length of more than 300 bp is suitable for the labeling with this kit. For labeling of DNA fragments shorter than 300 bp, it is recommended to use MEGALABEL (Cat.#6070), the kit for 5'-end labeling. This kit is designed to use 10 ng - 1 g of template DNA. DNAs embedded in low melting temperature agarose gels can be used directly in the reaction without the removal of agarose. SeaPlaque** GTG Agarose or NuSieve**GTG Agarose is suitable for this purpose. The procedure is as follows. 1) fter agarose gel electrophoresis, excise a slice of the gel containing the target DNA A fragment. 2) Add 3 volumes of distilled sterilized water to the agarose fragment. 3) Melt the agarose at 65. 4) The solution corresponding to 25 ng of DNA can be used directly in the reaction as template DNA. ** Trademarks of Lonza *2 This kit is designed for use of 1.85 MBq (50 Ci) [ -32P]dCTP (~ 111 TBq (3,000 Ci) /mmol), but35S]- or [3H]-labeled dCTP can also be used. When labeling with labeled dATP, use a [ solution of dGTP, dCTP, and dTTP (0.2 mM each) instead of the dNTP mixture included in the kit. *3 A probe with sufficient specific radioactivity can be obtained after 2-3 minutes of incubation, but the highest specific radioactivity is obtained after 10-20 minutes incubation which also can be extended for an overnight without significant loss of activity. (Fig. 2)

Figure 2. (The result was obtained by using 25 ng of DNA-Hin d III as the template. )
III. The effect of the amount of template DNA
mount of template DNA (ng) A 100 1,9 S 0.68 x 109 0.77 x 108 pecific radioactivity of probe (dpm/ g) 3.0 x 10 1.9 x 10 DNA Hin d III digest was labeled with 1.85 MBq (50 32P]dCTP (111 TBq Ci) of [ (3,000 Ci)/mmol), according to the recommended procedure. IV. Measurement of incorporation by DE81-filter binding assays 1) Dilute a small volume of the reaction mixture 20-fold with TE buffer or distilled sterilized water. 2) Spot 3 l of the diluted mixture, in duplicate, onto DE81 paper discs (Whatman) and dry. 3) Wash one DE81 disc six times with 100 ml of 5 % sodium phosphate, for 5 minutes each, twice with distilled water for 1 minute and twice with ethanol, and then dry. Measure the radioactivity with a liquid scintillation counter. 4) Measure the radioactivity of the other DE81 disc. 5) The incorporation yield and the specific activity of probe are calculated from the following formulas. (count of washed DE81 disc: cpm) Incorporation yield (%) = x 100 (count of unwashed DE81 disc: cpm) Ci added x 4 x 330 ng/nmol Theoretical yield (ng) = Specific activity of labeled dNTP ( Ci/nmol) If recommended amounts of labeled dCTP are used (50 Ci of [ -32P] dCTP with specific activity of 3,000 Ci/mmol), the theoretical yield of labeled probe will be 22 ng.
Total amount of probe (ng) = template DNA (ng) + incorporation yield x theoretical yield x 10-2 2.2 x 106 x Ci added x incorporation yield x 10-2 Specific radioactivity of probe (dpm/ g) = total amount of probe (ng) x 10-3
Also available from Takara are.
DNA labeling systems LaddermanTM Labeling Kit (Bca DNA polymerase / random-primed).. #6046 MEGALABELTM (DNA 5' labeling system)... #6070

NOTE:

This product is intended to be used for research purpose only. They are not to be used for drug or diag nostic purposes, nor are they intended for human use. They shall not to be used products as food, cos metics, or utensils, etc. Takara products may not be resold or transfered, modified for resale or transfer, or used to manufac ture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please call at +or contact from our website at www.takara-bio.com.
Phone: +81-77-543-7247 Fax: +81-77-543-9254

TaKaRa LA PCR Kit Ver.2.1

Table of Content

II. III. IV. V. VI.

Cat.# RR013A

v.0702
Description....2 Kit component...2 Storage....4 Reference.....4 Protocol A. Amplification of long DNA fragment...4. B. Amplification of DNA fragment with high GC rich contents..5 C. Electrophoresis....6 Application examples of LA PCR...7.
VII. Note....9 VIII. Q&A.... 10
Also available from Takara
PCR related products Cat. TaKaRa LA Taq TM.... #RR002 TaKaRa LA Taq TM Hot Start Version... #RR042 TaKaRa LA Taq TM with GC Buffer..#RR02AG Taq Antibody.... #9002 LA PCR Genome DNA Set... #9060. TaKaRa PCR Thermal Cycler DiceTM..#TP600/#TP650
http://www.takara-bio.com

TAKARA BIO INC.

I. Description:
LA PCR Kit Ver.2.1 The technology of the polymerase chain reaction (PCR) to amplify DNA sequences has now been wide spread in molecular genetics research, especially in genome analysis and sequencing studies. However, efficient amplification of DNA fragments greater than ~5 kb has been difficult, which limits some application of PCR. Incorporation of mismatched nucleotides is also one of the limitations to the PCR method. Production of specific PCR products has generally been limited to target templates of 5kb, at longest 10 kb. Recently, conditions such as DNA polymerase, reaction buffers, and thermal cycling profiles were identified for accurate amplification of longer DNA up to 40 kb. This LA (long and accurate) PCR technology* will extend the applications of PCR, especially it is expected to facillitate genome analysis in the various ways. TaKaRa LA PCR Kit Ver.2.1 includes the improved enzyme, TaKaRa LA Taq TM and buffers to achieve the amplification of long DNA fragments, such as human genomic DNA. This version 2.1 is a further improved kit of the old one (Ver.2) to work effectively for a highly-structured template as the buffers special for template with high GC contents are supplied in the kit. It is also available for the amplification of short fragments and other various kinds of template DNA. Including Mg2+ free buffer, this kit allows to choose optimal Mg2+ concentration of the reaction mixture according to a template DNA.
*U.S.Patent 5,436,149 for LA Technology is owned by TAKARA BIO INC.

II. Kit Components:

(50 reactions, for 50 l PCR)
1. TaKaRa LA Taq TM *1(5 units/ l)..125 units 2. dNTP Mixture*2(2.5 mM ea.)... 400 l 3.10x LA PCR Buffer II (25 mM Mg2+ plus).. 250 l 4.10X LA PCR Buffer II (Mg2+ free).. 250 l 5. MgCl2 (25 mM).... 500 l 6. Control Template...10 l (100 ng/ l genomic DNA derived from HL60 ) 7. Control Primer LA3*4 (10 M)...10 l. 8. Control Primer LA4*4 (10 M)...10 l. 9. -Hin d III digested MW Marker*4 (100 ng/ l).20 l 10. 2x GC Buffer I *3(5 mM Mg2+ plus)...1.25 ml 11. 2x GC Buffer II *3 (5 mM Mg2+ plus)..1.25 ml 12. Control Primer GC1*4 (10 M)...10 l 13. Control Primer GC2*4 (10 M)...10 l 1) TaKaRa LA Taq TM Concentration: 5 units/l Form: Supplied in 20 mM Tris-HCl (pH8.0), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Tween 20, 0.5% Nonidet P-40, 50% Glycerol Unit definition: One unit is the amount of the enzyme that will incorporate 10 nmol of dNTP into acid-insoluble products in 30 minutes at 74 , pH9.3, with activated soluble salmon sperm DNA as the template-primer. Reaction mixture for unit definition: 25 mM TAPS (pH9.3 at 25 ) 50 mM KCl 2 mM2 MgCl 1 mM 2-mercaptoethanol 200 M each dATP, dGTP, dTTP 100 M 32P]dCTP [ 0.25 mg/ml activated salmon sperm DNA Purity:Nicking activity, endonuclease and exonuclease activity were not detected after the incubation of 0.6 g of supercoiled pBR322 DNA, 0.6 g of DNA or 0.6 g of -Hin d III digest with 10 units of this enzyme for 1 hour at 74.
*2) dNTP Mixture (PCR Nucleotide Mix) Mixture of dNTP at the equal moles, ready for use in PCR without dilution. Concentration : 2.5 mM of each dNTP pH :7-9 Form : Solved in water (sodium salts) Purity : 98% for each dNTP *3) GC Buffers When a template may be highly complex structured or has high GC contents, it is recommended to use 2x GC Buffer I at first. In case that an amplified fragment cannot be obtained with 2x GC Buffer I, the result may be improved by using 2x GC Buffer II. *4) The sequence of Control Primers

Control Primer LA3: 5'-ACATGATTAGCAAAAGGGCCTAGCTTGGACTCAGA-3' Control Primer LA4: 5'-TGCACCTGCTCTGTGATTATGACTATCCCACAGTC-3'
Control Primer LA3 and LA4 will result 17.5 kbp amplified DNA fragment from Control Template.
Control Primer GC1: 5'-GGGAGGGGACCGGGGAACAGAG-3' Control Primer GC2: 5'-GAACAGTCCGTCACTTCACGTG-3'
Control Primer GC1 and GC2 will result 1,255 bp amplified DNA fragment with high GC contents from Control Template.
*4) -Hin d III digest MW Marker The range of size marker: 23,130, 9,416, 6,557, 4,361, 2,322, 2,027, 564, 125 bp. Reagents not supplied in the kit: 1. Agarose gel ex. SeaKem Gold Agarose (Cambrex Bioscience) 2. 6x Loading Buffer, containing 30% Glycerol and 0.25% Xylene Cyanol 3. Sterilized distilled water 4. Upstream and downstream primers Equipment required: 1. Authorized instruments for PCR TaKaRa PCR Thermal Cycler Dice (Cat.#TP600) 2. Microcentrifuge tubes (made of polypropylene) 3. Agarose gel electrophoresis apparatus 4. Microcentrifuge 5. Micropipets and pipette tips (autoclaved)
III. Storage: IV. Reference:
1) Barnes, W. M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216-2220. 2) Cheng, S. et al.(1994) Proc. Natl. Acad. Sci. USA 91, 5695-5699. 3) Cheng, S., Higuchi, R. and Stoneking, M. (1994) Nature Genet. 7, 350-351. 4) Cheng, S. et al. (1994) Nature 369, 684-685. 5) Saiki, R. K., Gelfand, D. H., Stoffel, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich, H. A. (1988) Science 239, 487-491. 6) Scharf, S. J., Horn, G.T. and Erlich, H. A. (1986) Science 233, 1076-1078. 7) Gyllensten, U. B. and Erlich, H. A. (1988) Proc. Natl. Acad. Sci. USA 85, 7652-7656. 8) Kawasaki, E. S., Clark, S. C., Coyne, M. Y., Smith, S. D., Champlin, R., White, O. N. and McCormick, F. P. (1988) Proc. Natl. Acad. Sci. USA 85, 5698-5702. 9) Lawyer, F. C., Stoffel, S., Saiki, R. K. Myambo, K. B., Drummond, R. and Gelfand, D. H. (1988) J. Biol. Chem. 264, 6427-6437. This kit includes DNA and primers for target sequence of DNA(20,707 bp).

V. Protocol:

A. Amplification of long DNA fragment This kit contains Control Primer LA3 and LA4 to amplify 17.5 kbp of Control Template (genome DNA from HL60). 1) Prepare the reaction mixture in reaction tube by combining the following reagents to the total volume of 50 l. [ In case of using 10 x LA PCR Buffer II (Mg2+ plus) ] Reagents Volume Final concentration TaKaRa LA Taq TM 0.5 l 2.5 units/50 l 10 x LA PCRTM Buffer II (Mg2+ plus) 5 l[ 1x ] dNTP Mixture 8 l each 400 M Template*1 X l <1g Primer 1*2 0.2 1.0 M (final conc.) 2 Primer 2* 0.2 1.0 M (final conc.) H2O yl Total 50 l [ In case of using 10 x LA PCR Buffer II (Mg2+ free) ] Reagents Volume Final concentration TM TaKaRa LA Taq 0.5 l 2.5 units/50 l 10 x LA PCRTM Buffer II (Mg2+ free) 5 l[ 1x ] 25 mM MgCll 2.5 mM dNTP Mixture 8 l each 400 M Template*1 X l <1g Primer 1*2 0.2 1.0 M (final conc.) Primer 2*2 0.2 1.0 M (final conc.) H2O yl Total 50 l *1 In case of control experiment, use 2 l (200 ng) of Control Template. *2 For Control Trmplate, use each 1 l of Control Primer LA3 and LA4.

2) If necessary, overlay mineral oil (approximately 50 l) to avoid the evaporation of the reaction mixture. * Some thermal cyclers do not require overlay of mineral oil. **When mineral oil needs to be removed completely from the reactant, extract the oil with chloroform (100 l). 3) Place tubes in Thermal Cycler. 4) Set the parameters to the following conditions. [TaKaRa PCR Thermal Cycler 480] 94C, 30 sec.*3 (denaturation) 68C, 15 min.*4 (annealing and extension) 30 cycles [TaKaRa PCR Thermal Cycler Dice, MP, TaKaRa PCR Thermal CyclerSP, GP, TaKaRa PCR Thermal Cycler PERSONAL] *3 *4 98C, 10 sec.*3 (denaturation) 68C, 15 min.*4 (annealing and extension) 30 cycles
Denaturation condition varies depending on an used thermal cycler and tube. It is recommended for 20 - 30 sec. at 94 or 5 - 10 sec. at 98. The annealing and extention time varies depending on the omplification size. The recommended time setting is 30 sec. - 1 min. per 1 kbp. In case of using Control Primer LA3 and LA4, the optimum time is 15 min.
Apply 5-10 l of PCR reactant to agarose gel electrophoresis, and verify the PCR products.
B: Amplification of DNA fragment with high GC rich contents This kit contains Control Primer GC1 and GC2 to amplify 1,255 bp of Control Template (genomic DNA from HL60) where the GC contents is 65%. 1) Prepare the reaction mixture in reaction tube by combining the following reagents to the total volume of 50 l. Reagents Volume Final concentration TaKaRa LA Taq TM 0.5 l 2.5 units/50 l 2 x GC Buffer I or 2 x GC Buffer II 25 l[ 1x ] dNTP Mixture 8 l each 400 M Template*1 X l <1g Primer 1*2 0.2 1.0 M (final conc.) Primer 2*2 0.2 1.0 M (final conc.) H2O yl Total 50 l *1 In case of control experiment, use 2 l (200 ng) of Control Template. *2 For Control Template, use each 1 l of Control Primer GC1 and GC2. 2) If necessary, overlay mineral oil (approximately 50 l) to avoid the evaporation of the reaction mixture. * Some thermal cyclers do not require overlay of mineral oil. *When mineral oil needs to be removed completely from the reactant, extract the oil with chloroform (100 l).
3) Place tubes in Thermal Cycler.
4) Set the parameters to the following conditions. 94 , 94 , 60 , 72 , 72 , 1 min. 30 sec.*sec. 30 cycles 2 min.*min.
5) Apply 5-10 l of PCR reactant to agarose gel electrophoresis, and verify the PCR products. *3 *4 C: Electrophoresis 1) Take 5~10 l of PCR amplified samples and add its 1/6 volume of 6x Loading Buffer. 2) Pipet the samples into the agarose gel slots, and run the gel. The conditions of agarose gel vary according to sizes of amplified DNA. 3) After electrophoresis is completed, stain gels by soaking in Ethidium Bromide solution(0.5 g/ml) for 30-60 min, or SYBR Green I (Cambrex). 4) Verify the bands of amplified DNA under UV illumination. In case of control experiment, the 17.5 kbp amplification product is obtained for PCR when the Control Primer LA3 and LA4 are used, the 1,225 bp amplification product is obtained when the Control Primer GC1 and GC2 are used. Denaturation condition varies depending on an used thermal cycler and tube. It is recommended for 20 - 30 sec. at 94 , or 5 - 10 sec. at 98. The extention time varies depending on the amplification size. The recommended time setting is one minute per 1 kbp. In case of using Control Primer GC1 and GC2, the optimum time is 2 min.

VI. Application examples of LA PCR: 1) Target : Reaction mixture: Reagents
Amplification of human genomic DNA -globin cluster region TPA gene region
Volume Final concentration
10x LA PCR Buffer II (Mg2+ plus) 5 l[1x] dNTP Mixture 8 l each 400 M Sense Primer (20 pmol/ l) 0.5 l 0.2 M Antisense Primer (20 pmol/ l) 0.5 l 0.2 M TaKaRa LA Taq TM 0.5 l 2.5 units/50 l Human genomic DNA (500 ng/ l)1 l 500 ng/50 l Sterlized distilled H2O 34.5 l Total 50 l *Overlay 50 l of Mineral oil. ( Some thermal cyclers do not require overlay of mineral oil.)
PCR condition: [Thermal Cycler480]
94 , 1 min. 94 , 30 sec. ** 68 , 15 min. (17.5 kbp, 21.5 kbp) 30 cycles or 20 min. (27 kbp) 72 , 10 min. ** In case of using TaKaRa Thermal Cycler MP, SP, GP, Dice or PERSONAL, set 98 , 10 sec.

Lane:

PCR products size
1: -Hin d III Marker 2:17.5kb ( -globin cluster) 3: 21.5kb ( -globin cluster) 4: 27 kb (TPA gene) 5: High Molecular Weight DNA Marker (Invitrogen) 5 l/50 l PCR 0.4% Agarose gel electrophoresis
2) Amplification of E.coli genomic DNA (> 18 kbp) The concentraiton of template DNA is 100 ng/50 l. The other reaction conditions (composition of reaction mixture and PCR condition) are the same as of the Amplication example 1).
Lane: 1: 2: 3: 4: 5: 6: PCR products size -Hin d III Marker 20 kb 28 kb 30 kb 38 kb High Molecular Weight DNA Marker (GIBCO BRL)
5 l/ 50 l PCR 0.4% Agarose gel electrophoresis
3) Amplification of E.coli genomic DNA ( 18 kbp) The compositions of reaction mixture are the same as of the Amplication example 2). PCR condition: [Thermal Cycler480] 94 , 1 min. 94 , 30 sec. ** 68 , 3 min. (2 kb, 4 kb), or 5 min. (6 kb, 8 kb), or 30 cycles 15 min. (10 kb, 18 kb) 72 , 10 min. ** In case of using TaKaRa Thermal Cycler MP,SP or PERSONAL, set 98 , 10 sec.
1: -Hin d III Marker 2: 2 kb 3: 4 kb 4: 6 kb 5: 8 kb 6:10 kb 7:18 kb 8: -Hin d III Marker 5 l/ 50 l PCR 1% Agarose gel electrophoresis
4) Amplification of Huntington's disease gene (high GC contents) The amplification performances were compared among the conventional LA PCR Buffer and newly developed GC Buffer I, II. Target : Huntington's disease (HD) gene IT 15 CAG repeat region 262 bp - GC contents : 73% 358 bp - GC contents : 71.5 % PCR condition: [Thermal Cycler MP]
94 , 1 min. 94 , 30 sec. 60 , 30 sec. 30 cycles 72 , 1 min. 72 , 5 min.

Buffer

M:100 bp DNA Ladder 1:10x LA PCR Buffer 2: 2x GC Buffer I 3: 2x GC Buffer II 8 l/ 50 l PCR 3% Agarose gel electrophoresis (Nusieve 3:1 Agarose gel (Cambrex)

VII. Note:

1) Mix well the kit components by pipetting or converting tubes up and down after melting completely at the room temperature~ 37. It is recommended to avoid mixing vigorously by vortex mixer. Especially when mixing 10X LA PCR Buffer and TakaRa LA Taq TM, mix carefully and gently to avoid producing bubbles or inactivation of enzymes. Each component should be kept on the ice until used. 2) Mix gently the reaction mixture by pipetting before starting reactions. In this case, never use vortex mixer. 3) Reactiom mixture should be prepared to the total volume of 10~50 l when amplifying long target DNA. 4) High specific primers should be used to obtain long PCR products. The use of primers as long as 30~35 mers is advisable for high specificity. 5) Template DNA should be highly purified as possible. The volume of template DNA for amplification of long DNA is 4~5 times of general PCR.

1. PCR Optimization

VIII. Q&A:
Optimal reaction conditions vary according to amplified sizes, reaction volumes, and types of an used thermal cycler. i) Cycle numbers Set the optimum cycle number around 25-30 cycles by considering the quantity or complexity of template DNA and the length of amplified DNA fragments. Less cycling may not generate enough amplified product, while over cycling may produce a diffuse smear upon electrophoresis. ii) Denaturation conditions When using thin-wall type PCR tubes, denaturation conditions recommended are either at 98 for 10 sec. or at 94 for 20 sec. When using normal PCR tubes, denaturation conditions are recommended to be either at 94 for 30 sec. or at 98 for 20 sec. A denaturation time that is too short or a denaturation temperature that is too low may cause either diffuse smearing upon electrophoresis or poor amplification efficiency. A denaturation time that is too long or a denaturation temperature that is too high may generate no identifiable product. iii) Conditions for Annealing and Extension Determine the optimum annealing temperature experimentally by varying temperatures in 2 increments over a range of 45-68. As TaKaRa LA Taq TM shows sufficient activity at 60-68 , Shuttle PCR (Two Temperature PCR) can be conducted by setting the anneal-extension temperature within this range. To carry out the combined annealing extension at 68 (two step PCR), the recommended time setting is 30 sec. to 1 min. per 1 kb. When temperature is set below 68 , longer time will be required. An annealing temperature that is too high generates no amplification products, while a temperature that is too low enhances non-specific reactions. An extension time that is too short generates no amplification products or dominantly non-specific, short products, while too long extension time causes diffusely smeared electrophoresis bands.

2. Primer preparation

Specificity of primers is very important for the generation of longer DNA amplification products. For example, if it is highly specific, even 20-mer primers can generate DNA fragments in length of greater than 20 kb. If possible, prepare primers according to the following criteria. 1) The primer length should be 30 to 35 bases. 2) The difference between the optimum annealing temperature of paired primers should be within 2-3. 3) Choose primers whose GC contents is around 50-60%. 4) Avoid primer sequence which form hairpin loops or primer-dimers, especially at the 3' end. 5) Primers including inosin within its sequence should be avoided. The optimal concentration will range from 0.1 M to 1.0 M. At a lower than optimum concentration, amplification yield may be poor. On the other hand, at a higher concentration, non-specific reactions may outperform primer-specific amplifications. In ordinary practice, primer concentrations can be determined depending on the characteristics and amounts of template DNA: low concentrations are recommended either for highly complex DNA such as human genomic DNA, or for high concentrations of template DNA, while high concentrations are preferred for low complexity templates such as plasmid DNA, or for limiting amounts of template DNA.

3. Primer Concentration

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4. Enzyme Amount

Although it is recommended to use 2.5 units of TaKaRa LA Taq TM in a 50 l reaction for which TaKaRa LA PCR Kit Ver.2.1 is designed, you may change the amount used for the optimum reaction condition. The following factors should be taken into consideration; the quantity or complexity of template DNA and the length of amplified DNA fragment. In case of excess enzyme, non-specific reactions may occur which may result in a diffuse smear upon electrophoresis. The efficiency of amplification may be diminished when the enzyme concentration is low. When you perform PCR to generate DNA fragments longer than 10 kb, the preparation of intact (free of nicks) and fully purified template samples through repeated extraction/purification is essential. Samples which are prepared by only lysing cells with either heat treatment or protease digestion are not appropriate for LA PCR. Dr. GenTLE (from whole Blood) Cat.#9081) allows the simple extraction of DNA suitable for LA PCR from 100 l of blood. The following describes the recommended preparation protocols for human genomic DNA and E.coli genomic DNA. i) Protocol for the preparation of Human Genomic DNA 1. Harvest HL60 cells (from 15 petri dishes for about 1.5 x 109 cells) and wash twice with 0.7% NaCl. 2. Suspend cells with 15 ml of 10 mM Tris-HCl (pH8.3) in a polycarbonate centrifuge tube. 3. Add 150 l of 10 mg/ml Proteinase K (Takara Cat.#9033) and 150 l of 10% SDS into cell suspension for the lysis. 4. Incubate the cell and reagent mix at 60 for one hour and subsequently at 37 for additional 16 hours. 5. Add 15 ml of phenol saturated with 1 M Tris-HCl (pH8.0), into the tube and mix gently for 15 min with repeated inversions of the tube. 6. Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 min. 7. Transfer an aqueous phase to a new tube. 8. Add 15 ml of phenol/chloroform/isoamylalcohol (25:24:1, saturated with TE buffer, pH9.0) into the tube and gently mix it up and down in the same way as the step 5 for 15 min. 9. Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 min. 10. Transfer an aqueous phase to a new tube. 11. Add 15 ml of chloroform/isoamylalcohol (24:1) into the tube and gently mix it up and down in the same way as the step 5 for 15 min. 12. Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 min. 13. Transfer an aqueuos phase to a new tube. 14. Add 1.5 ml of 3 M Sodium Acetate (pH5.2) and 30 ml of 99.5% ethanol and mix gently. 15. Collect DNA around a thin glass stick and wash it with 80% ethanol. 16. Dry DNA. 17. Dissolve DNA with 10 ml of TE buffer by letting it stand at 4 overnight. 18. Add 100 l of 10 mg/ml RNase A into the DNA solution and incubate at 37 for one hour. 19. Add 10 ml of phenol/chloroform/isoamylalcohol (25:24:1) and mix gently for 5 min. 20. Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 min. 21. Transfer the aqueous phase into a new tube. 22. Add 10 ml of chloroform/isoamylalcohol (24:1) and mix gently for 5 min. 23. Centrifuge at 9,000 rpm (approx. 6,000 g) at room temperature for 10 min. 24. Transfer the aqueous phase into a new tube. 25. Add 1 ml of 3 M Sodium Acetate (pH5.2) and 20 ml of 99.5% ethanol into the tube and mix gently. 26. Collect DNA around a thin glass stick. 27. Wash DNA with 80% ethanol. 28. Dry DNA. 29. Dissolve DNA in 2 ml of TE buffer and bring DNA concentration to 0.5 mg/ml.

5. Template DNA

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ii) Preparation Protocol for E.coli Genomic DNA 1. Grow E.coli overnight in two 1-liter flasks containing 200 ml of L-broth. 2. Harvest cells and suspend them in 40 ml of 50 mM Tris-HCl (pH8.0) containing 50 mM EDTA. 3. Deep-freeze cells at -80 for 30 min. 4. Add 4 ml of 10 mg/ml lysozyme solution in 0.25 M Tris-HCl (pH8.0) to frozen cells and then thaw them at room temperature with occasional mixing. 5. Leave thawed cells on ice for 45 min. 6. Add 8 ml of STEP (0.5% SDS, 50 mM Tris-HCl (pH8.0), 0.4 M EDTA and 1 mg/ml Proteinase K) and incubate at 50 for one hour. 7. Add 45 ml of TE (10 mM Tris-HCl (pH8.3) and 1 mM EDTA). 8. Then add 96 ml of phenol/chloroform/isoamylalcohol (25:24:1, saturated with TE buffer pH9.0) into the tube and gently mix by repeated inversions for 5 min. 9. Centrifuge at 5,000 rpm (approx. 3,000 g) at room temperature for 15 min. 10. Transfer the aqueous phase to a new tube. 11. Add 96 ml of chloroform/isoamylalcohol (24:1) into the tube and gently mix it up and down in the same way as in step 8 for 5 min. 12. Centrifuge at 5,000 rpm (approx. 3,000 g) at room temperaturefor 15 min. 13. Transfer the aqueous phase to a new tube. 14. Add 9 ml of 3 M Sodium Acetate (pH5.2) and 225 ml of 99.5% ethanol and mix gently. 15. Collect DNA around a thin glass stick and wash it with 80% ethanol. 16. Dry DNA. 17. Dissolve DNA in 20 ml of TE buffer by letting it stand at 4 overnight. 18. Add 10 l of 10 mg/ml RNase A into the DNA solution and incubate at 37 for 30 min. 19. Add 20 ml of phenol/chloroform/isoamylalcohol (25:24:1, saturated with TE buffer, pH9.0) and mix gently for 5 min. in the same way as the step 8. 20. Centrifuge at 10,000 rpm (approx. 6,500 g) at room temperature for 15 min. 21. Transfer the aqueous phase into a new tube. 22. Add 20 ml of chloroform/isoamylalcohol (24:1) and mix gently for 5 min. 23. Centrifuge at 10,000 rpm (approx. 6,500 g) at room temperature for 10 min. 24. Transfer the aqueous phase into a new tube. 25. Add 2 ml of 3 M Sodium Acetate (pH5.2) and 50 ml of 99.5% ethanol into the tube and mix gently. 26. Collect DNA around a thin glass stick. 27. Wash DNA with 80% ethanol. 28. Dry DNA. 29. Dissolve DNA in 20 ml of TE buffer by letting it stand at 4 overnight. (DNA concentration will be around 0.1 mg/ml.) Genomic DNAs of human and E.coli which were prepared through the above protocols are commercially available from TaKaRa for the use as control template in LA PCR. LA PCR Genome DNA Set (Size: ea. 20 reactions).. Cat.#9060 (including both human genomic DNA and E.coli genomic DNA) Recommended DNA amount in LA PCR human genomic DNA: 0.1 - 1 g / 50 l PCR E.coli genomic DNA :10 - 100 ng/ 50 l PCR phage DNA: 0.5 - 2.5 ng/ 50 l PCR

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6. Is it possible to directly subject phage particle to LA PCR?
The lysates of approximately 106-107 PFU which was obtained by heating 99 for 10 min. can generate DNA fragments in length around 8 kbp. 7. Is it possible to directly subject cell lysates obtained by either heat treatment (98 for 2 min.) or protease digestion from either mammalian cells or E.coli cells to LA PCR amplification? - In case of E.coli , it is possible. Lysates of heat-treatment alone can generate DNA fragments in length around 10 kbp. (* E.coli cells were cultured in L-broth medium at 37 overnight. Two l of the cell culture was used for 50 l.) - In case of human cell (cultured ), the extent of purification significantly affects the length of extension in PCR amplification. Heat treatment alone has a limit of several hundred bases, while double treatment with heat and protease may exceed 1-2 kbp. For longer length amplifications, it is recommended to use purified DNA prepared with our standard protocols described in 5.
8. Amplified products are entirely smeared upon electrophoresis. Possible Causes Comments and suggestions
Too much enzyme amount Reduce the enzyme amount in 0.5 units steps. Too short denaturation time Increase the denaturation time by increments of 5 sec. Too low denaturation temperature Raise the denaturation temperature by 0.5 intervals. Too low dNTP concentration Too long extension time Too many number of PCR cycles Too much template amount Increase the dNTP concentration in by increments of 50 M steps. Shorten the extension time by decrements of 1 min. Reduce the number of cycles in steps of 2 cycles. Reduce the template amount by decrements of 20% of the previous one.

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9. Multiple, nonspecific amplified products upon electrophoresis Possible Causes

Comments and suggestions

Too high primer concentration Decrease the primer concentration in steps of 0.1 M. Enhance the specificity of primers by changing the complementary region of the template or by preparing longer primers up to 30- 35 mer. Reduce the enzyme amount in 0.5 units steps. Reduce the number of cycles in steps of 2 cycles. Raise the annealing temperature by 2 intervals. Use Hot Start method to avoid thisoccurs. phenomenon during heating from room temperature to the denaturation temperature (94-98.) Increase the extension time by increments of 1 min. Raise the denaturation temperature by 0.5 intervals and extend the time by increments of 5 sec. Reduce the template amount by decrements of 20% of the previous one.

Poor primer design Too much enzyme amount Too many number of PCR cycles Too low annealing temperature Nonspecific annealing of primers Too short extension time Poor denaturation Too much template amount
10. Does TaKaRa LA Taq TM produce a sticky end (3'-A overhang) like TaKaRa Taq TM ? Is it possible to use TA cloning? Yes, TaKaRa LA Taq TM generates DNA fragments with 3'-A overhang that can be cloned into T-vectors almost at the same efficiency as TaKaRa Taq TM does. Therefore, the amplified products can be applied to TA-cloning. The efficiency will be lowered in case of cloning long PCR products (>5 kbp) into T-Vector. 11. What is the appropriate agarose gel to use for the electrophoresis of longer DNA fragments generated with LA PCR? It is recommended to use 0.4% SeaKemTM Gold Agarose (Cambrex).

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NOTICE TO PURCHASER: LIMITED LICENSE
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No right under any other patent claim (such as the patented 5' Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. LA PCR Technology : U,S,Patent 5,436,146 for LA Technology is owned by TAKARA BIO INC.

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NOTE: This product is intended to be used for research purpose only. They are not to be used for drug or diagnostic purposes, nor are they intended for human use. They shall not to be used products as food, cosmetics, or utensils, etc. Takara products may not be resold or transfered, modified for resale or transfer, or used to manufacture commercial products without written approval from TAKARA BIO INC. If you require licenses for other use, please call at +or contact from our website at www. takara-bio.com.