Flex-C Cloning Kit - Appleton Woods Ltd
Contents
1. Use media plates with appropriate too much antibiotics amount of correct antibiotics Suboptimal PCR product Use different purification method Inhibitory contaminants Both the PCR product and the from PCR product or linearized vector should be purified linearized vector Transform with too much Do not add more than 10 of reaction mixture reaction mixture to 50ul of competent cells Too much reaction mixture inhibits the transformation Colonies do not Incomplete linearization of Make sure the vector is completely have insert vector digested and purify by gel DNA recovery method Re digest and purify the vector if necessary Contamination of other If insert was amplified from a plasmids with same antibiotic plasmid circular DNA may have resistance carried through purification and contaminated the cloning reaction Users are recommended to gel purify the PCR product or the linearizing template DNA Colonies contain Insert contain unspecific If the PCR product is not a single incorrect insert sequences distinct band gel purify the correct insert www vivantechnologies com info vivantechnoogies com2. eTo Any linearized vector B Preparation of Linearized Vector A linearized vector can be generated by PCR or restriction enzyme digestion single or double digestion A complete digested vector is critical in order to achieve high cloning efficiency Users are recommended to purify the linearized vector by gel DNA recovery method Due to the differences in digestion efficiency different restriction enzymes will generate different amounts of background Users are encouraged to perform double digestion to reduce the opportunity of self ligation of the vector User may increase the incubation time 4 hours up to overnight depending on the enzymes properties for a more complete digestion reaction Users can test the background by transforming 10 50ng of purified and linearized vector into competent cells If the background is high perform the digestion using more restriction enzymes or prolong the incubation period C Primer Design Primer design is very crucial for a successful cloning reaction Users can clone two or more fragments into any linearized vector as long as they share 10 homologous bases at each end Primers should be designed as per the following 1 The 5 end of the primer MUST contain 10 bases homologous to 10 bases at one end of the DNA fragment to which it will join not including the restriction site That can be the vector or another insert 2 The 3 end of the primer MUST contain the gene speci
3. everse Primer Example 5 Forward primer Kpn 5 A TTC GAG CTC GGT ACC XXX XXX 3 5 GCC AGT GAA TTC GAG CTC GGT AC G GCA TGC AAG CTT GGC 3 3 CGG TCA CTT AAG CTC GAG C AC GTC CGT ACG TTC GAA CCG 5 E 3 XXX XXX GAC GTC CGT ACG TTC G 5S PstI 3 Reverse Primer Figure 2 Primer design with restriction site of restriction enzymes generating 3 overhangs IlI Linearized Vector with Blunt Ends Forward Primer append with restriction site and gene specific sequence 5 NNNNNNNNNN _ gt L y 12345678910 5 NNNNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNNNN 3 3 NNNNNNNNNNNNNNNN 5 3 NNNNNNNNNNNNNNNNN 5 10987654321 4 NNNNNNNNNN 5 Reverse Primer Example 5 Forward primer Din 5 T ACC GCA TCA GGC GCC XXX XXX 3 5 GGA GAA AAT ACC GCA TCA GGC GCC ATT CGC CAT TCA GGC TGC 3 3 CCT CTT TTA TGG CGT AGT CCG CGG TAA GCG GTA AGT CCG ACG 5 3 XXX XXX CCG CGG TAA GCG GTA A 5 Din 3 Reverse Primer Figure 3 Primer design with restriction site of restriction enzymes generating blunt ends Primers above are designed with restriction site of restriction enzymes generating 5 overhang figure 1 3 overhang figure 2 and blunt end figure 3 Restriction sites are highlighted in red and X represent bases corresponding to the specific gene
4. fic sequence It can be designed using 18 20 bases with GC content between 40 60 3 Melting temperature is calculated based on the 3 gene specific sequence of the primer instead of the entire primer sequence 4 Avoid complementary sequences within the primers and between primers Please refer to the examples below for primer design I Linearized Vector with 5 Overhang Forward Primer append with restriction site and gene specific sequence 5 NNNNNNNNNN gt ica 12345678910 5 NNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNNNNNN 3 3 NNNNNNNNNNNNNNNNNN 5 3 NNNNNNNNNNNNNNN 5 10987654321 E lt _NNNNNNNNNN 5 Reverse Primer Example 5 Forward primer BamH 5 C GGT ACC CGG GGA TCC XXX XXX 3 eye 5 GAG CTC GGT ACC CGG G AG CTT GGC GTA ATC ATG GTC 3 3 CTC GAG CCA TGG GCC CCT AG A CCG CAT TAG TAC CAG 5 3 XXX XXX TTC GAA CCG CAT TAG T 5 Hind Ill 3 Reverse Primer Figure 1 Primer design with restriction site of restriction enzymes generating 5 overhangs Il Linearized Vector with 3 Overhang Forward Primer append with restriction site and gene specific sequence 5 NNNNNNNNNN 12345678910 5 NNNNNNNNNNNNNNNNNN 3 5 NNNNNNNNNNNNNNN 3 3 NNNNNNNNNNNNNN 5 3 NNNNNNNNNNNNNNNNNNN 5 10987654321 z lt _ NNNNNNNNNN 5 R
5. ree system gt Joining multiple fragments at once gt Broad PCR size up to 10kb gt Good for 5 overhangs 3 overhangs blunt ends gt Precise insertion at a desired orientation gt High Efficiency with gt 95 positive clones gt Multiple applications adding adaptor linker and tag before or after the insert mutation generation gene synthesis gt High throughput application Components MEPLO2 MEPLO2 S Flex C Cloning Enzyme 20 app 5 app Taq DNA Polymerase 500u 50u 10X ViBuffer A 2ml imi 10X ViBuffer S imli 1ml 50mM MgCle Iml 1ml 10mM dNTPs mix 0 25ml 0 025ml Nuclease free Water 1ml iml Storage Store at 20 C Taq DNA Polymerase Storage Buffer 20mM Tris HCI pH8 0 at 22 C 100mM KCI 0 5 Tween TM 20 0 5 Nonidet P40 0 1mM EDTA 1mM DTT and 50 glycerol 10X ViBuffer A 500mM KCI 100mM Tris HCl pH9 2 at 20 C and 0 1 Triton M X 100 10X ViBuffer S 160mM NH4 2S04 500mM Tris HCl pH9 2 at 22 C 17 5mM MgCl2 and 0 1 Triton X 100 Additional items to be supplied by user Gene specific primers with 10bp homolog to vector ends Linearised vector Competent cells LB plates containing appropriate antibiotic A Protocol Overview Amplify your gene of interest Linearize any vector Flex C Enzyme Design gene specific a primers with i 10bp homolog to vector ends EE Hm Single tube reaction a A 10 min room temperature 7 10 min on ice gt w w gt oy H oo Transformation
6. sequences The 10 homologous bases are referring to the vector sequences that are adjacent to the restriction sites D PCR Product Consideration 1 We recommend users to use Taq DNA Polymerase or other high fidelity polymerase for amplification of gene of interest 2 Purify the PCR product by gel DNA recovery method to remove undesired bands E Cloning Procedure 1 Set up a reaction mixture as follows Linearized vector 60 200ng 2ul Purified PCR product 20 200ng ul Flex C Enzyme Mix 2ul Nuclease free water top up to 10ul Total Volume 10ul Recommended molar ratio for vector and insert is 1 3 In general 1 5 to 3 1 of vector and insert ratio will produce good results 2 Incubate at room temperature for 10 minutes and on ice for 10 minutes 3 Proceed to transformation Commercial competent cells are recommended 4 Plate the cells onto LB plates containing appropriate antibiotic for the cloning vector Incubate all the plates for overnight at 37 C 5 Perform PCR screening or restriction enzyme digestion to determine the presence of insert F Trouble shooting Few or no colonies Poor quality competent cells Test transformation efficiency obtainedfrom the using supercoiled plasmid DNA transformation gt 108 colonies per ug of supercoiled DNA is expected Incorrect primer sequence Check primer sequences to make it contains 10 homologous bases to the ends of the vector Wrong antibiotics used or with
7. vivantis Flex C Cloning Kit MEPLO2 20 applications MEPLO2 S 5 applications User Manual Description Flex C Cloning Kit is a highly efficient rapid and easy to use PCR cloning kit The Flex C Enzyme allows direct cloning of any PCR fragments into any linearized expression vector at any site in a single 20 minute reaction The application protocol is simple The PCR fragments can be generated by PCR Polymerases for eg Taq DNA Polymerase with primers that are designed to have at least 10 bases of homology with the vector at their linear ends No additional treatment of the PCR fragment is required such as restriction digestion ligation phosphorylation or blunt end polishing The linearized vector can be generated by PCR or restriction enzymes single or double digestion Flex C Enzyme joins PCR fragments and linearized vectors accurately and efficiently by recognizing the 10bp overlap at their ends This method allows cloning of multiple fragments into a single vector in a single reaction without subcloning to create fusion proteins to delete and replace DNA sequences or to insert point mutations The Flex C Cloning Kit is highly efficient with a 95 insert rate The Flex C Cloning Kit also includes Vivantis Taq DNA Polymerase and reaction buffers as well as dNTPs for subsequent PCR screening of clones Key Features gt Clone any insert at any site within any vector gt Restriction enzyme phosphatase and ligase f
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