Microsoft word - blk-101.doc

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Instruction manual Blunting high 0810 F0990K Blunting high [1] Introduction
[2] Components
[3] Protocol
[4] Troubleshooting
[5] References


CAUTION
All reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe
general laboratory precautions and follow safety guidelines while using this kit.

JAPAN
TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
www.toyobo.co.jp/e/bio
Blunting high is a kit that produces a blunt end at the DNA terminus, and allows for its use in a subsequent ligation step using KOD DNA polymerase1) and DNA ligase reagents, respectively. 5’ over hang
over han ed
5’-N-N-
5’-N-N-N
3’-N-N-
3’-N-N-N
olymerase
3’ over hang
over han ed
5’-N-N-
5’-N-N-N
3’-N-N-
3’-N-N-N
Fig. 1. Principle of Blunting by KOD DNA polymerase
- The blunting step makes use of KOD DNA polymerase that exhibits 5’J 3’ polymerase - The blunting step is completed in 2 min. - A highly efficient premixed ligation reagent, “Ligation high” is included in this kit. - A control reaction can be performed using the control DNA provided. This kit includes the following components for 20 reactions. All reagents should be Notes: - Ligation high is a highly efficient premixed ligation reagent - The control DNA is pUC18 plasmid digested by EcoRI and Sph I. Following blunting and subsequent ligation, the circular control plasmid DNA generates E. coli colonies on - 10 x Blunting Buffer contains 1.2 M Tris-HCl (pH 8.0 at 25 °C), 100 mM KCl, 15 mM MgCl2, 60 mM (NH4)2SO4, 2 mM dNTPs, 1% TritonX-100, and 0.01% BSA. TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
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1. Blunting
Control reaction
* KOD (2.5 U/μl) should be added last. This enzyme exhibits strong 3’ J5’ exonuclease activity in the absence of dNTPs. For this kit, DNA should be dissolved in distilled water or TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). However, in the following cases, DNA solutions may be used undiluted, directly with this kit. - Restriction enzyme-treated DNA
Restriction enzyme-treated solutions using High, Medium, Low or Tris-Acetate buffer can be directly used according to the above protocol.
- PCR products of Taq DNA polymerase
PCR solution (amplified by Taq-based polymerase) *10 x Blunting Buffer should not be added. *If the concentration of dNTPs in the PCR solution is less than 0.2 mM, add TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
www.toyobo.co.jp/e/bio
This kit can process 10 pmol blunt DNA per reaction. 10 μg pUC18 (linearized; 2.7 kb) corresponds to 10 pmol. The maximum volume of DNA solution is 8 μl. When preparing the reaction solution, KOD (2.5 U/μl) should be added last. KOD DNA polymerase exhibits strong 3’ J5’ exonuclease activity in the absence of The reaction is completed within 2 min. The reaction time can be prolonged up to 2. Ligation
Control reaction
Blunted Control DNA (from [3] 1. Blunting)* - The reacted solution can be directly used for transfection experiments using E. - For the control experiment, E. coli colonies can be observed on ampicillin plates. The colony number in the control experiment using the blunted control DNA will be at least 20 times greater than that using the not blunt control DNA. - KOD DNA polymerase should not be inactivated prior to the ligation step because this polymerase is hardly active at 16 °C. - The reacted solution from the blunting reaction can be used directly in a ligation reaction, but excessive carry-over of the solution reduces ligation efficiency. In such cases, prolong the reaction time up to overnight, or purify the blunted TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.
www.toyobo.co.jp/e/bio
Solution
Addition of too much reacted mixture from the blunting reaction to the ligation reaction tends to Decrease the volume of the reacted mixture from the blunting reaction or exchange the buffer of the reacted mixture obtained from the blunting reaction to a low salt solution (e.g. distilled water or TE KOD DNA polymerase degrades DNA from the 3’ end by its 3’J5’ exonuclease activity in the absence of dNTPs. KOD DNA polymerase must be added last. 1) Takagi M, Nishioka M, Kakihara H, Kitabayashi M, Inoue H, Kawakami B, Oka M, and Imanaka T., Appl Environ Microbiol., 63: 4504-10 (1997) TOYOBO CO., LTD. TOYOBO Bio-Technology, CO., LTD. Tel(81)-6-6348-3888 Tel(86)-21-58794900.4140 www.toyobo.co.jp/e/bio FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

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