Then use a recombinase we use Exnase II in my lab to put the constructs and the vector together in a homologous recombination step before transforming the product into the desired competent host. 2.
Nov 03 2020 The obtained feruloyl esterase genes were extracted from the gel after electrophoresis. The pET 22b vector was digested by NdeI and XhoI and then purified. To ligate the gene into pET 22b the vector and fragment were mixed with the molar ratio of 2 1 and the Exnase II was used to activate the homologous recombination.
For a starch granule marker full length granule bound starch synthase I StGBSS without a stop codon was amplified from E3 cDNA with specific primers and cloned into pJCV55 obtained by restriction with BglII using Exnase II Vazyme . Primer sequences are shown in Supplementary Table S2.
with Exnase II ClonExpress II Vazyme to generate pLP12‐ ∆pslA and was further confirmed by primers pLP‐UF and pLP‐UR. DH5α and PA47 wild‐type strains were transformed with this plasmid. Deletion of pslA was confirmed by PCR using primers PslA‐MF1 and PslA‐ MR2. Detailed information about the strains and primers is shown in
Dec 15 2018 For vector construction full length StPHYF and truncated forms of StPHYF and StPHYB were amplified with specific primers Table S1 and separately cloned into the EcoRI and SalI sites of the pGBKT7 vector using Exnase II Vazyme .
So we need to use Exnase II C214 Vazyme to cut the overlapped DNA segment and then recombined to circled DNA S and S D614G construct 1. Exanse II 0.5ul recombination Exanse II enzyme 1ul 5X buffer 3.5ul DMT digested product=total 5 ul
5 CE II buffer 2 μl 2 μl Exnase II 1 μl 1 μl ddH 2 O to 10μl to 10 μl to 10 μl to 10 μl a. X\Y represent vector and insert DNA amount calculated according to the formula linear vector 0.02 vector base pair number ng 0.015 pmol Insert DNA 0.06 insert DNA base pair number ng 0.045 pmol . b.
The highly optimized reaction buffer and the enhanced recombinase Exnase II significantly improve the recombination efficiency of cloning and the tolerance to impurities making it possible for linearized vectors and inserts to be directly used for recombination cloning without purification greatly simplifying The experimental steps.
Nov 16 2016 The protein marker recombinase exnase II high fidelity enzyme DNA polymerase and E. coli strains DH5α and BL21 DE3 were purchased from Vazyme Biotech Nanjing China . A low molecular weight protein ladder and restriction endonuclease were
We next purified the exact size of S D614G PCR product by gel extraction then we used Exnase II C214 Vazyme to make the linearized product circled. Then circled S D614G plasmids were transformed into DH5α competent cells single clones were
Exnase II 1 ddH2O 1 Total volume 10 1.7 Transformation of recombinant plasmid After the recombination reaction the system is transferred into E. coli DH5α competent cells. The experimental operations are as follows 1 Remove DH5α competent cells from 80 ℃ refrigerator place it on ice and thaw it to
Exnase II . 2 μl . Vector DNA . Volume calculated by CE Design . Insert DNA . Volume calculated by CE Design . H2O . Volume calculated Incubate at 37 C for 30 min. Bacterial Transformation Protocol Take chemically competent DH5α cells out of 80 C and immediately place it on ice. Extract 20 μL competent cells and add altogether 2 μL of
plasmids using Exnase II for 30min at 37 C. The Zhang et al. BMC Biotechnology 2019 19 59 Page 2 of 12. reaction mixture consisted of 200ng Dpn I digestion product 4μL of 5 CE II Buffer 2μL of Exnase II and ddH 2Oupto20μL. These plasmids were sequenced
Exnase€ I l Mutated Vector Non Methylated Transformation Fig 1 A. 43 Exnase© rninRIJÃJ Ill. Ill. Mutation Point A Mutation Point B A Forward Primer Primer B Forward Primer B Reverse Primer Original Vector Methylated Methylated Templates Digestion Dpn l Linear Amplification Non Methy ated Homologous Recombination Exnasee Il
Exonuclease III . E. coli. Double stranded DNA specific exonuclease. Initiates at the 3 termini of linear double stranded DNA with 5 overhangs or blunt ends and 3 overhangs containing less than four bases. Initiates at nicked sites in double stranded DNA. Catalyzes the removal of nucleotides from linear or nicked double stranded DNA in
Nov 01 2017 with Exnase II Vazyme China . The vector was then transferred into E. coli for confirmation. Plasmids. pBRAd4 A3R7 1 and pBRAd4 A3R7 2 were generated with the same method. The successful
ClonExpress II One Step Cloning Kit Vazyme Biotech Co. Ltd . Typically 20 μL of a mixture containing 4 μL of 5 CE II Buffer 2 μL of Exnase II 50 ng linear plasmid 100 ng PCR fragment and ddH2O was incubated at 37oC for 30 min and the resulting solution was used to transform chemically competent E. coli DH5α cells. Colony PCR and
72˚C for 8 min. Following enzyme digestion Exnase ii Vazyme Biotech co. td. 1l µl using loncexpress ii one Step c loning kit Vazyme Biotech co. l td. and sequencing the Pcr product was cloned into the i/KpnXhoi sites of the GV230 expression vector. The recombinant GV230 septin7 plasmid was confirmed by endonuclease digestion and
binant enzyme Exnase II. The resulting recombinant plasmid was transformed into competent E. coli DH5α cells and transformed cells were spread on MH plates 12 μg/mL TC 0.3 D Glucose . Positive recombinant clones containing the AB fragment were screened using primers pLP UF/pLP UR. After screening the positive
May 12 2020 In vitro recombination of heterologous modules crtZ and crtW. To further improve the yield of astaxanthin in yQDD001 in vitro recombination was used to rearrange the heterologous genes of astaxanthin in vitro e/LoxP was a widely used site specific DNA recombination system derived from bacteriophage P1.
Recombinases. Cre Recombinase NEB #M0298 is a Type I topoisomerase from bacteriophage P1 that catalyzes the site specific recombination of DNA between loxP sites 1 . The loxP recognition element is a 34 base pair bp sequence comprised of two 13 bp inverted repeats flanking an 8 bp spacer region which confers directionality 3 .
Nov 01 2019 The tcpA constructs were obtained from SC09 genomic DNA by recombinase Exnase II ClonExpress II Vazyme and then cloned in pCMV Tag 2B. 2.9. Co immunoprecipitations CO IP Rainbow trout head kidney macrophages were infected with the wild type SC09 for 12 h. Cells were washed twice in ice cold PBS harvested and cell lysis buffer was added.
Dec 01 2020 The AB fragment was purified and ligated with the suicide vector pLP12 using the recombinant enzyme Exnase II. The resulting recombinant plasmid was transformed into competent E. coli DH5α cells and transformed cells were spread on MH plates 12 μg/mL TC 0.3 D Glucose . Positive recombinant clones containing the AB fragment were screened
In addition Exnase II is compatible with the reaction systems of restriction enzyme digestion and PCR which means that both the digestion products of vector and the PCR products of insert can be directly used for recombination without purification
Feb 03 2020 After pre denaturation at 95 C for 30 s thermal cycling was performed at 95 C for 15 s 70 C for 15 s and 72 C for 3 min 30 cycles followed by a final extension at 72 C for 5 min. DpnI 10 U was then added and incubated at 37 C for 1 or 2 h for template digestion and Exnase II was added and incubated at 37 C for 30 min for
The authors describe the use of Exnase II in restriction enzyme free cloning of inserts into plasmids. They show that their method leads to a higher frequency of scarless plasmids then the Exnase cloning or Two step PCR ligase method. Due to the poor English it took me a while before I was able to understand what the message of the paper was.
Jun 28 2019 The resultant PCR products was recovered and digested with BamHI and HindШ and inserted into pQE80L using Exnase II. The recombinant plasmids were transformed into E. coli JM109 and plated on the LB agar plate. Each mono colony was picked up and inoculated into 96 deep well plate of LB medium and cultivated at 37 C and 180 rpm overnight to
The highly optimized reaction buffer and the enhanced recombinase Exnase II significantly improve the recombination efficiency of cloning and the tolerance to impurities making it possible for linearized vectors and inserts to be directly used for recombination cloning without purification greatly simplifying The experimental steps.
Jun 09 2020 Then the linearized vector purified PCR product buffer and enhanced recombinase Exnase II were mixed and incubated for 30 min yielding the recombinant vectors. The recombinant vectors were transformed into A. baumannii ATCC 17978 A. pittii LMG1035 and A1254 by electroporation. Quantitative Reverse Transcription PCR
Apr 09 2020 The purified PCR product for the fiber gene was then ligated with the purified linear pGEX 6P 1 Exnase II ClonExpress II kit Vazyme Biotech and transformed into regular chemically competent cells as described previously. 17 Positive recombinants were confirmed by
Exnase II 1 ddH2O 1 Total volume 10 1.7 Transformation of recombinant plasmid After the recombination reaction the system is transferred into E. coli DH5α competent cells. The experimental operations are as follows 1 Remove DH5α competent cells from 80 ℃ refrigerator place it on ice and thaw it to
The stir 2 sequences from SC09 genomic DNA were ligated into pCMV Tag 2B using Exnase II ClonExpress II Vazyme Nanjing china . 4.9. Co Immunoprecipitations CO IP Rainbow trout head kidney macrophages were infected with wild type SC09 for 12 h. Cells were washed twice in ice cold PBS harvested and cell lysate was added.
We previously constructed a PAO1 PA2146 knockout strain ΔPA2146 briefly the upstream and downstream fragments of PA2146 were amplified and the products were then fused by overlapping PCR and the fused fragment was cloned into pLP12 KnoGen Biotech Guangzhou China using recombinant enzyme Exnase II ClonExpress II Vazyme to generate
Following enzyme digestion Exnase II 1 µl using ClonExpress II One Step Cloning kit Vazyme Biotech Co. Ltd. Nanjing China and sequencing the PCR product was cloned into the Xho I Kpn I sites of the GV230 expression vector.
Dec 01 2020 A CCAAT box transcription factor SlNFY10 is a candidate regulator of ascorbic acid biosynthesis. GME1 and GME2 are two homologous genes
Add 1ul Exnase II Ligase and 2ul CE II Buffer 5x . 3 Incubate at 16 C for 1 hour or at 4 C for overnight .
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