Cloning,! 免付費客服專線 TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) U040114

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1 2015 Cloning, Cloning,! U TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線

2 (Genetic Cloning) Cloning, (Molecular Cloning), (Restriction enzyme;re) DNA, DNA (DNA ligase) (Vector), ( E.coli ) Good Science, TaKaRa Clontech, TaKaRa Clontech, Cloning Get the Right Clone,! Seamless Cloning In-Fusion Cloning HD Plus System 效應 2 Traditional Cloning DNA Ligation Kits DNA Ligation 01

3 效應 2 比一比 In-Fusion Cloning Kits 效應 2 DNA Ligation Kits DNA Inserts TA cloning Blunt-end cloning Long-insert cloning Cloning Simple constructs Complex constructs Simple constructs clone insert 5 1 clone construct 15 (5 inserts/reaction) N/A Site-directed mutagenesis X High-throughput applications Subcloning X ( MCS, clone ) X Cloning X 02

4 In-Fusion Cloning HD Plus System Cloning,

5 Seamless Cloning Learn More In-Fusion Cloning HD Plus System DNA, >95% Single-insert cloning Multiple-insert cloning Large-insert cloning Adding small tags Direct cloning into large vectors (up to 46-kb cosmids) Site-directed mutagenesis (single or multiple deletions, insertions, or substitutions) No Subcloning No Ligation One-step Directional Cloning Low-Background Screen Fewer Colonies, In-Fusion? 15bp, 1 3 PCR reaction 4 PCR : a) Cloning Enhancer b) Gel extraction 5 In-Fusion : In-Fusion DNA, Insert vector, 2 15 bp PCR primer 5, In-Fusion primer 6 In-Fusion, 1 1 : a) b) PCR 04

6 Seamless Cloning In-Fusion Cloning HD Plus System,! Stellar TM Competent Cells,> 5 x 10 8 cfu/µg PCR Product Treatment PCR NucleoSpin Gel and PCR Clean Up Kit Any PCR -amplified DNA Most applications Cloning Enhancer Single PCR Fragment of expected size Convenient for High-Throughput Cloning 4 CloneAmpHiFi PCR Premix (5sec/ Kb) In-Fusion HD Enzyme Premix 3'->5' exonuclease, cloning vector PCR insert 15 bp overhangs 15 bp annealing clone, Subcloning clone! Kit Components : Product Cat No Size Cloning enhancer NucleoSpin Stellar competent cells CloneAmpHiFi PCR premix Rxns In-Fusion HD Cloning Plus Rxns rxns In-Fusion HD Cloning Plus CE rxns 05

7 Seamless Cloning In-Fusion Cloning HD Plus System! Interstellar?! In-Fusion with Stellar! The In-Fusion HD Cloning System now includes high-quality Stellar Competent Cells, which consistently outperform other cells when cloning small and large DNA fragments. 15 kb insert! 4 inserts! Ten out of ten colonies contain the correct insert (100% efficiency) when cloning fragments as large as 15 kb (results confirmed by colony PCR screening). Using In-Fusion HD system, cloning up to four fragments simultaneously is as easy as cloning a single fragment. Just combine the PCR fragments with appropriate complementary ends and a linear vector, then incubate. In-Fusion mutagenesis : point mutation, deletion insertion 1. : Choose the vector you want to modify and envision your final, mutated construct. 2. : Design inverse primers that overlap each other by 15 bp at their 5'ends and incorporate your desired deletion, substitution, or addition. 3. In-Fusion : Using an inverse PCR protocol, amplify the vector with your new primers. 4. : Recover your mutant from the Stellar cells the following day. Application Note 06

8 Seamless Cloning In-Fusion Cloning HD Plus System Online Tools for In-Fusion Cloning Primer Design Tool Design primers for In- Fusion PCR cloning. Enter the DNA sequences of both vector and insert, along with your linearization method for the vector. Compatible with Firefox and Chrome only. PCR Primers Clontech, Cloning Vector 15bp overlap DNA Insert Primers * Primer Design Tutorial! Molar Ratio Calculator Calculate the optimal amounts of vector and insert for an In-Fusion cloning reaction. Just enter the sizes of both vector and insert, in base pairs. Molar Ratio In-Fusion Vector Insert (ng) Simulate Your Construct SnapGene software allows easy planning and visualization of molecular biology procedures, including In- Fusion cloning. SnapGene In-Fusion Cloning, Fuse DNA, SnapGene primers,! 07

9 Seamless Cloning In-Fusion Cloning HD Plus System Jun Yang Instructor, University of Texas Medical Branch Cloning into Lentiviral Vectors with In-Fusion HD Cloning Plus In-Fusion Cloning was used to overcome the problem of restriction site availability in lentiviral vectors. Five different inserts were directly cloned into the final vector with a success rate nearing 100%. In my experience, the In -Fusion method is very convenient, reliable, and timesaving. Ti-Yu Lin Graduate Student, University of Wisconsin In-Fusion HD Cloning Plus: A Fast, Accurate Alternative to Ligation Where a previous ligation-based cloning experiment had failed, In-Fusion Cloning generated positive clones with a success rate of 80%, on the first attempt. Ansul Lokdarshi Graduate Research Assistant, University of Knoxville, TN Fast, Direct Cloning into Large Vectors with In-Fusion HD Cloning Plus A large binary vector for in planta localization studies of YFP- tagged proteins was completed with just one cloning reaction, and a success rate of 100%., In-Fusion Cloning kit size (120bp, 200bp, 323bp, 832bp) >5000 bp vector In-Fusion Cloning kit Cloning,,,, In-Fusion Cloning kit overlapping 15bp,! transformation colony, fusion,,,! 08

10 Ligation Kits, : Insert Reaction speed Type of ends (blunt, A-overhang or sticky) Ligation Kits High-Throughput... DNA Ligation Kits 09

11 DNA Ligation 效應 2 Traditional Cloning DNA Ligation Kits Learn More Ligation Made Easy! DNA Ligation Kit, Mighty Mix, 5! 10 µl, sample cohesive T/A Cloning Blunt end cloning One Solution Premix,, DNA Takara DNA Ligation Kit, Mighty Mix Blunt-Ended Ligation TA-Vector Ligation! Blunt-Ended Ligation Efficiency Comparison with Takara's DNA Ligation Kit, Mighty Mix and Three Competitors. A : Promega's, LigaFast, B : BioLine's, QUICK-STICK and C: NEB, Quick Ligation Kit. Each ligation reaction contained 25 fmol of BAPtreated puc118-hinc II vector and 75 fmol of a 500 bp insert. TA-Vector Ligation Efficiency Comparison of Takara's DNA Ligation Kit, Mighty Mix Against Three Competitors : A : Promega, LigaFast, B : BioLine, QUICK-STICK and C: NEB, Quick Ligation Kit. Each ligation reaction contained 13 fmol of a pt7 Blue T-Vector and 39 fmol of a 2.08 kb PCR product. Product Cat No Size Components DNA Ligation Kit, Mighty Mix Rxns Ligation Mix 5 x150 µl 10

12 效應 2 Traditional Cloning DNA Ligation Kits TaKaRa DNA Ligation Kit LONG BAC DNA library ligation 10 kb inser ligation ~ T/A Cloning Blunt end cloning 18 kb control insert Takara DNA Ligation Kit, LONG 18kb Ligation 50,! Comparison of Ligation Efficiency with Various DNA Ligation Sits. Several size DNA fragments, 2kb, 4kb, 10kb and 18kb digested by Hin d III were ligated into the cloning vector puc118/ Hind III/BAP using Ligation Kit Long (Lig LONG) and the other commercially available DNA ligation kits. Ligation reactants were transformed into E.coli DH5α cells and grown overnight on LBamp plates at 37. Product Cat No Size Components DNA Ligation Kit, LONG Rxns DNA Ligase 50 µl 10X LONG Ligation Buffer 300 µl Control Insert DNA/Hind III (18kb) 50 µl Control Vector (puc 8/HindIII/BAP) 10 µl dh 2 O 1 ml TaKaRa Ligation! DNA Ligation Kit, Mighty Mix TaKaRa DNA Ligation Kit LONG Speed TA cloning Blunt-end cloning Sticky-end cloning Small volume High throughput Long inserts & BAC library construction 11

13 Cat. # Product Size In-Fusion Cloning Rxns In-Fusion HD Cloning Plus ( Liquid system, includes: In-Fusion HD Enzyme Premix, CloneAmp HiFi PCR Premix, Stellar Competent Cells, and NucleoSpin Gel and PCR Clean-Up kit ) 50 Rxns Rxns Rxns In-Fusion HD Cloning Plus CE ( Liquid system, includes: In-Fusion HD Enzyme Premix, CloneAmp HiFi PCR Premix, Stellar Competent Cells, and Cloning Enhancer ) 50 Rxns Rxns DNA Ligation Kits 6023 DNA Ligation Kit, Mighty Mix Rxns 6024 TaKaRa DNA Ligation Kit LONG 50 Rxns 12

14 20154/30 TEL (02) TEL (05) TEL (03) TEL (06) TEL (04) TEL (07) 免付費客服專線