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Summary of Contents for Bioneer AccuRapid K-7170
- Page 1 AccuRapid ™ TA Cloning Kit Cat. No. K-7170 © Copyright 2022 BIONEER Corporation. All rights reserved.
- Page 2 AccuRapid™ TA Cloning Kit User Guide K-7170 Version No.: 2 (2024-07-15) Please read all the information in booklet before using the unit BIONEER Corporation 8-11, Munpyeongseo-ro, Daedeok-gu, Daejeon 34302, Republic of Korea Tel: 1588-9788 Email: sales@bioneer.co.kr www.bioneer.com...
- Page 3 Material Safety Data Sheet (MSDS). Warranty and Liability All BIONEER products undergo extensive Quality Control testing and validation. BIONEER guarantees quality during the warranty period as specified, when following the appropriate protocol as supplied with the product. It is the responsibility of the purchaser to determine the suitability of the product for its particular use.
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Page 5: Table Of Contents
AccuRapid™ TA Cloning Kit Contents Product Information …………………………………………………………………………… Components ………………………………………………………………………………….. Storage ……………………………………………………………………………………….. Specifications ………………………………………………………………………………… Introduction ……………………………………………………………………………………… Product Description …………………………………………………………………………. pBHA-T Vector ……………………………………………………………………………….. Experimental Procedures ……………………………………………………………………… Materials to be Prepared by Users ………………………………………………………… Preparation of Insert ………………………………………………………………………… Ligation Reaction …………………………………………………………………………….. Transformation ……………………………………………………………………………….. Results Analysis …………………………………………………………………………….. -
Page 6: Product Information
AccuRapid™ 2X Reaction buffer. Therefore, it is recommended to aliquot the solution and store each tube for single reaction. Specifications AccuRapid™ TA Cloning Kit Reaction time 15 min Reaction temperature 25°C Need for PCR/Gel purification Recommendation Selection marker Ampicillin BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 7: Introduction
BIONEER's AccuRapid™ TA Cloning Kit is a product that can easily and quickly clone the PCR product amplified with a DNA polymerase such as Taq DNA Polymerase that can add dA to the 3' end into a linearized pBHA-T vector. -
Page 8: Pbha-T Vector
It is used to analyze sequences to confirm if the gene of interest is inserted in the plasmid or to transcribe RNA from inserted sequence in vitro. 5. BamH I restriction site It is used to digest the inserted DNA from the recombinant plasmid. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 9 Vector Map pBHA-T vector Position (Bases) M13 Reverse primer site 1-17 LacZα gene 13-441 SP6 promoter 35-53 T7 promoter 236-254 M13 Forward primer site 261-277 Ampicillin resistance gene 1,003-1,863 pUC ori (high copy number) 2,034-2,622 BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com...
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Page 10: Experimental Procedures
• Chemically or electrocompetent cell • IPTG (Cat. No. C-8001) • X-Gal (Cat. No. C-8002) • LB agar plate with ampicillin • LB broth or SOC media • M13 Forward primer, M13 Reverse primer BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 11: Preparation Of Insert
4. After electrophoresis of the amplified insert, gel elution is performed with AccuPrep ® PCR/Gel Purification Kit (Cat. No. K-3038). * Note: Using an unpurified PCR product can significantly decrease the cloning efficiency, so it is recommended to purify the target DNA band through gel purification. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 12: Ligation Reaction
* Note: Commercially available elution buffer contains EDTA, which can reduce ligation efficiency. It is recommended to use the elution buffer (EA buffer) without EDTA of BIONEER’s AccuPrep ® PCR/Gel Purification Kit or distilled water. Ligation Reaction 1. Thaw pBHA-T vector and AccuRapid™ 2X Reaction buffer on ice before ligation reaction. -
Page 13: Transformation
6. After the incubation, centrifuge at 3,000 rpm for 5 min, remove the supernatant, add 200 μl of LB broth, and gently resuspend the cell pellet by tapping. 7. Transfer to preheated LB agar plate (included antibiotics) in step 5, and spread completely. 8. Incubate overnight at 37°C in an incubator. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 14: Results Analysis
* Expected size of DNA in experimental group: 249 bp † + size of insert DNA † Length between M13 Forward primer and M13 Reverse primer * Expected size of DNA in control group: 649 bp BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 15 ATT TAG GTG ACA CTA TAG M13F(-40) GTT TTC CCA GTC ACG AC M13R(-40) CAG GAA ACA GCT ATG AC M13F(-20) GTA AAA CGA CGG CCA GT M13R(-20) GCG GAT AAC AAT TTC ACA CAG G BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com...
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Page 16: Troubleshooting
Low molar ratio of insert DNA and vector ratio of 5 times or more for ligation reaction. High salt concentrations may inhibit the ligation Inhibited ligation reaction reaction. It is recommended to minimize the amount (volume) of PCR product. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 17 4. Most colonies are blue or light blue. Cause Solution Check whether both IPTG and X-Gal are No X-Gal or IPTG in LB agar plates. properly added to in LB agar plates. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com...
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Page 18: Ordering Information
T4 DNA Ligase E-3061 IPTG C-8001 X-Gal C-8002 Agarose, 100 g C-9100 50X TAE ,500 ml C-9004 GreenStar™ Nucleic Acid Staining Solution I C-9036 AllInOneCycler™ PCR System A-2041 Agaro-Power™ System A-7020 DUALED Blue/White Transilluminator A-6020 BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 19: Explanation Of Symbols
AccuRapid™ TA Cloning Kit Explanation of Symbols Consult Batch Catalog Caution Instructions Code Number For Use Contains Research Temperature Sufficient for Manufacturer Limitation <n> tests Only Use-by Date BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 21 AccuRapid™ TA Cloning Kit 사 용 설 명 서 K-7170 Version No.: 2 (2024-07-15) 사용 전, 사용설명서에 있는 모든 내용을 정독하시길 바랍니다. ㈜ 바이오니아 대전광역시 대덕구 문평서로 8-11 Tel: 1588-9788 Email: sales@bioneer.co.kr www.bioneer.com...
- Page 23 사용 목적 AccuRapid™ TA Cloning Kit는 연구용 제품으로 연구 목적으로만 사용할 수 있습니다. 사용자는 국가 및 사용 용도에 따라 권한 취득이 필요할 수 있습니다. 안전경고 및 주의사항 자극적이거나 유해한 물질을 다루는 경우 적절한 보호장비를 착용하시기 바랍니다. 실험복, 보호장갑, 보호안경 등의 사용을 적극 권장합니다. 자세한 정보는 물질안전보건자료 (MSDS)를 확인하시기...
- Page 25 AccuRapid™ TA Cloning Kit 목차 제품 정보 …………………………………………………………………………………………………….. 제품 구성 …………………………………………………………………………………………………………………... 보관법 ………………………………………………………………………………………………………………………… 18 제품 사양 …………………………………………………………………………………………………………………... 개요 …………………………………………………………………………………………………………….. 제품 개요 ………………………………………………………………………………………………………………….. pBHA-T Vector …………………………………………………………………………………………………………... 실험 방법 …………………………………………………………………………………………………….. 사용자 준비 사항 ……………………………………………………………………………………………………… Insert 준비 ………………………………………………………………………………………………………………… Ligation 반응 …………………………………………………………………………………………………………….. 형질전환 …………………………………………………………………………………………………………………….. 결과...
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Page 27: 제품 정보
AccuRapid™ 2X Reaction buffer 를 반복적으로 얼리고 녹일 경우, cloning 효율이 저하될 수 있습니다. 따라서, 1회 반응분을 따로 분주하여 보관하는 것을 권장합니다. 제품 사양 AccuRapid™ TA Cloning Kit 반응 시간 15분 반응 온도 25°C PCR/Gel purification 필요성 권장 사항 선별 마커 Ampicillin BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 28: 제품 개요
포함되어 있어, 결과 분석이나 다른 vector로의 cloning을 쉽게 합니다. BIONEER의 AccuRapid™ TA Cloning Kit는 3’ 말단에 (dA)를 첨가할 수 있는 Taq DNA Polymerase와 같은 DNA polymerase 로부터 증폭된 PCR 산물을 선형화 된 pBHA-T vector에 쉽고 빠르게 cloning 할... -
Page 29: Pbha-T Vector
4. T7 promoter and SP6 promoter 삽입하고자 하는 DNA 서열이 plasmid에 포함되어 있는지 서열을 분석하거나 삽입한 서열로부터 RNA를 in vitro transcription할 때 사용합니다. 5. BamH I restriction site 삽입된 DNA 를 재조합 plasmid 로부터 잘라내는데 사용합니다. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 30 Vector Map 위치 (Bases) pBHA-T vector M13 Reverse primer site 1~17 LacZα gene 13~441 SP6 promoter 35~53 T7 promoter 236~254 M13 Forward primer site 261~277 Ampicillin resistance gene 1,003~1,863 pUC ori (high copy number) 2,034~2,622 BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com...
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Page 31: 실험 방법
• Chemically or electrocompetent cell • IPTG (Cat. No. C-8001) • X-Gal (Cat. No. C-8002) • Ampicillin 이 포함된 LB agar plate • LB broth 혹은 SOC 배지 • M13 Forward primer, M13 Reverse primer BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 32: Insert 준비
PCR/Gel Purification Kit (Cat. No. K-3038)로 gel elution 합니다. * 참고: 정제되지 않은 PCR product 를 이용할 경우, 효율이 현저히 떨어질 수 있어, gel purification 을 통해 원하는 DNA band 를 정제하여 사용하는 것을 권장합니다. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 33: Ligation 반응
4. 반응용액을 25°C 에서 최소 15분간 반응을 진행합니다. * 참고: 15분 반응만으로 충분한 colony 를 얻을 수 있지만, cloning 효율을 증가시키고 싶다면 ligation 반응을 1시간까지 늘리는 것을 권장합니다. 5. 반응이 완료된 후, 반응용액은 형질전환을 수행할 때까지 얼음 또는 -20°C 에서 보관합니다. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 34: 형질전환
6. 배양이 끝난 뒤, 3,000 rpm 으로 5분간 원심 분리하여 상층액을 제거하고 LB broth 200 μl 를 넣어, cell pellet 을 tapping 하여 부드럽게 풀어줍니다. 7. 5번에서 예열한 항생제가 포함된 LB agar plate 로 옮겨, 완전히 스며들도록 spreading 합니다. 8. 37°C incubator 에서 overnight 배양합니다. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 35: 결과 확인
4. 전기영동을 수행하여 insert DNA 삽입 유무를 확인합니다. * 실험군 예상 DNA band 크기: 249 bp + insert DNA 의 크기 ┗ M13 Forward primer 와 M13 Reverse primer 사이의 길이 * 대조군 예상 DNA band 크기: 649 bp BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 36 ATT TAG GTG ACA CTA TAG M13F(-40) GTT TTC CCA GTC ACG AC M13R(-40) CAG GAA ACA GCT ATG AC M13F(-20) GTA AAA CGA CGG CCA GT M13R(-20) GCG GAT AAC AAT TTC ACA CAG G BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com...
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Page 37: 문제 해결
염농도가 높은 경우 ligation 반응을 저하시킬 수 Ligation 반응이 제대로 이루어지지 않은 경우. 있습니다. PCR product의 사용량(부피)을 최소화 하는 것을 권장합니다. Host cell이 blue/white screening에 적합하지 않 Host cell의 유전형을 확인하시어 lacZ 유전자에 은 경우. mutation이 있는지 확인하시길 바랍니다. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 38 유전자의 reading frame이 손상되어 활성을 나타 내지 않게 됩니다. 4. 백색 colony만 형성됨. 원인 해결 방법 LB agar plate에 X-Gal과 IPTG를 적절하게 첨가 LB agar plate에 X-Gal 또는 IPTG가 없는 경우. 하였는지 확인하시기 바랍니다. BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com...
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Page 39: 주문정보
T4 DNA Ligase E-3061 IPTG C-8001 X-Gal C-8002 Agarose, 100 g C-9100 50X TAE ,500 ml C-9004 GreenStar™ Nucleic Acid Staining Solution I C-9036 AllInOneCycler™ PCR System A-2041 Agaro-Power™ System A-7020 DUALED Blue/White Transilluminator A-6020 BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... -
Page 40: 기호설명
AccuRapid™ TA Cloning Kit 기호설명 Consult Batch Catalog Caution Instructions Code Number For Use Contains Research Temperature Sufficient for Manufacturer Limitation <n> tests Only Use-by Date BQ-042-101-01 Revision : 4 (2022-02-14) www.bioneer.com... - Page 41 BQ-042-101-01 © Copyright 2022 BIONEER Corporation. All rights reserved. Revision : 4 (2022-02-14) www.bioneer.com...
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