pbad totpo ta expression kit and clonejet pcr cloning kit: Tender Category: Miscellaneous tenders : Tender Location: maharashtra : Tender Value: Refer Document: Tender Competition: Domestic competitive bidding: Bid Opening Date: 24 Aug 2022: Doc Purchase End Date : 24 Aug 2022 : Publication: 04 Aug 2022: FirstTender Id : 220804224820 CloneJET PCR Cloning Kit is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Because you lose some DNA during the gel purification step, it is important to digest . It is ideal for phosphorylated or non-phosphorylated DNA fragments. The plasmid sequence shows forward and reverse primers (electropherograms) that correspond to the E. histolytica rRNA gene sequence. Each rxn: . the PCR reaction. Digest your DNA: Set up restriction digests for your PCR product and recipient plasmid. The Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive-selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Refer to the competent cell manual for transformation protocol. The Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. (CloneJET PCR cloning kit, Fermentas), and E. coli NEB5 . Clone any blunt or sticky-end DNA fragment whether DNA fragments are phosphorylated or non-phosphorylated. The CloneJET PCR Cloning Kit is also compatible with all common E.coli laboratory strains. As a result, only bacterial cells with recombinant plasmids are able to form colonies. As far as I know, there are at least five different kits for cloning blunt-end PCR product. PCR product cloning with InsTAclone PCR Cloning Kit. Expression of recombinant -galactosidase add . 1) PCR using the pJET primers (often included in the kit, if you use the Thermo Scientific CloneJET PCR Cloning kit). The kit works well with both phosphorylated and non-phosphorylated DNA fragments. 1. Specific PCR products of <1 kb appearing as one discrete band on the gel can be used for ligation directly from PCR reaction mixture without any purification. Recommendations Thoroughly mix every vial before use. Blunt ends are needed to insert into pJET vector (or you can do a blunting rxn). protocols. Thoroughly mix every vial before use. CloneJET PCR cloning kit: Thermo Fisher: Cat#K1232: DIG labeling kit: Merck/Roche: Cat#11277073910: NucleoSpin Gel and PCR Clean-up kit: Macherey-Nagel: Cat#740609.50: According to kit self ligated vector should not grow . The Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. Thermo fisher CloneJET PCR Cloning Kit. The pDrive Cloning Vector (see figure "pDrive . Extract genomic DNA from the cell . recommended for use with the CloneJET PCR Cloning Kit. 1 l of T4 DNA ligase (5 U/l) was added to the mixture . All common laboratory E.coli strains can be directly transformed with the ligation product. We are using pJET1.2 vector (Thermo CLoneJET PCR Cloning kit) for cloning 1400bp fragment. Transformation efficiency of competent 4 IMPORTANT NOTES Include final extension step in the PCR cycling protocol to ensure efficient 3'-dA tailing of the PCR product. It allows simple and quick cloning of any PCR amplicon, whether the amplification reactions are performed with proofreading . This PCR Cloning Kit contains an optimized Cloning Mix containing a proprietary ligation enhancer and a linearized vector that uses a novel mechanism for background colony suppression to give a low background. Follow the Protocol provided by the manufacturer but with the following modifications: 1) use the 1.6 volume of magnetic beads to sample ratio (i.e. Any blunt or sticky-end DNA fragment can be cloned. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. 2.4. Incorrect PCR amplicon was used during cloning: Optimize the PCR conditions; Gel purify the correct PCR fragment ; Internal recognition site was present: Use NEBcutter to analyze insert sequence for presence of an internal recognition site; DNA fragment of interest is toxic to the cells: Incubate plates at lower temperature (25 - 30C) Use a DNA isolation protocol. Thermo Scientific CloneJET PCR Cloning Kit, also available with DH10B cells ( K123120 ), is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. PCR cloning with low/no background A 500 bp PCR product incubated with the linearized vector in a 3:1 ratio according to recommended protocol. 2. Any other blunt or sticky-end DNA . Bioz Stars score: 86/100, based on 1 PubMed citations. Specific PCR products of <1 kb appearing as one discrete band on the gel can be used for ligation directly from PCR reaction mixture without any purification. This protocol is optimized for monocot tissue based on protocols from Jackson and Kellogg labs (Jackson, 1991; Jackson et al., . A nayz e 4-6 c oies f r thp r sd n f DNA t u g Transformation of competent E.c oli cells with the ligation mixture can be performed using f l wing meth ds: different transformation methods (Table 2 and 3). The CloneJET PCR Cloning Kit is compatible with all PCR buffers supplied by Thermo Scientific. It is ideal for phosphorylated or non-phosphorylated DNA fragments. Any other blunt. Any other blunt or sticky-end DNA fragment can be cloned. Ligation into the positive selection vector takes only five minutes, yielding more than 99% recombinant clones. Recircularised pJET1.2/blunt vector molecules lacking an insert express a lethal . Add to Cart. The Thermo Scientific CloneJET PCR Cloning Kit is an advanced positive selection system for high-efficiency cloning of PCR products generated with any thermostable . Thermo fisher Zero Blunt TOPO PCR . There is no problem of self-ligation because this vector contain self killing gene. Gene transcripts were PCR-amplified with the Q5 HF DNA polymerase (cat no M0491, New England Biolabs, Ipswich, MA, USA) using primers listed in Tables S1 and S2, sub-cloned using the 'CloneJet PCR Cloning Kit' (cat no K1231, Thermo Fisher Scientific), and Sanger-sequenced at Eurofins Genomics (Ebersberg, Germany). . Clones were analyzed by PCR to verify the insertion of the amplicon into the pJET1.2/blunt vector. according to suppliers, protocols. Thermo Scientific CloneJET PCR Cloning Kit is a versatile positive selection system for high-efficiency cloning of PCR products generated with any thermostable DNA polymerase. 86. The CloneJET PCR cloning kit contains a ready-to-use positive selection cloning vector pJET1.2/blunt. This is one of the best kit for initial cloning. The PCR product is now ready for restriction digestion. . Russell,Molecular Cloning: A Laboratory Manual, (2001) by J Sambrook andD W Add To MetaCart. Perform a colony PCR with a standard Taq polymerase on at least 5 colonies. Gel-analyze the PCR product for specificity and yield before cloning. Therefore, in such cases, the protocol for each strain needs to be optimized. The Thermo Scientific CloneJET PCR Cloning Kit is a versatile, advanced positive selection system for high-efficiency cloning of PCR products. you can use fresh colony (Colony PCR), or you can do a plasmid extraction . Sorted by: Results 1 - 10 of 19. If the PCR product has to be used for cloning using Thermo Scientific CloneJET PCR Cloning Kit (#K1231), the final extension step can be omitted . Any other blunt or sticky-end DNA fragment can be cloned. PROTOCOL | NOVEMBER 25, 2021 Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells . according to the protocol of Aukrust and Blom [25]. Isolate your PCR product from the rest of the PCR reaction using a kit, such as the QIAquick PCR Purification Kit. Gel-analyze the PCR product for specificity and yield before cloning. Gel-analyze the PCR product for specificity and yield before . Clonejet Pcr Cloning Kit, supplied by Thermo Fisher, used in various techniques. In addition, there is a possibility that parts of the genomic DNA sequence vary depending on the strain of E. gracilis used. The following protocol was used in PCR: an initial denaturing step at 94 C for 3 min, followed by 35 cycles of 94 C for 1 min, 57 C for 30 s and 72 C for 70s; and a final extension time step at 72 C for 5 min. 2 l of reaction was transformed into provided NEB 10-beta Competent E. coli and 1/20th of the outgrowth was plated. Apart from that this is highly efficient kit and give approx. The Thermo Scientific CloneJET PCR Cloning Kit is a versatile, advanced positive selection system for high-efficiency cloning of PCR products. To clone the PCR products, we used a CloneJET PCR cloning kit (Thermo Fisher Scientific). The final extension step prolonged to 20-30 minutes generally yields 3-4 fold higher numbers of recombinant clones. Transform chemically competent TOP10 cells with the ligation product and plate on carbenicillin selection plates. The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. Before designing the target sequence, it is also . CloneJET PCR Cloning Kit The Thermo Scientific CloneJET PCR Cloning Kit can be used with PCR products generated by any DNA polymerase, therefore supporting both blunt- and sticky-end fragments from 6 bp to 10 kb long. For sequence validation, the PCR product was subcloned using CloneJET PCR cloning kit (Thermo Scientific). Next 10 . Transformation of competent E.coli cells with the ligation mixture can be performed using different transformation methods (Table 2 and 3). CloneJET PCR Cloning Kit: ThermoFisher: K1231: NovaBlue Competent Cells: Merck: 69825: TRI Reagent Solution: ThermoFisher: AM9738: . Transformation Analysis of recombinant clones The CloneJET PCR C loning Kit is compatible with all common E.coli laboratory strains. Ligation mixtures (2 L) were used to transform E.coli DH10B cells. 85. We have blunted our insert following kit protocol. 3. All common laboratory E.coli strains can be directly transformed with the ligation product. Bioluminescence-Sensing Assay for Microbial Growth Recognition by Heba Ramadan Eed , Nora S Abdel-Kader , Mahmoud Helmy , El Tahan , Tianhong Dai , Rehab Amin . Left plate serves as the control, with vector backbone only, right plate contains PCR . ZERO BIAS - scores, article reviews, protocol conditions and more Ligation mixtures (2 L) were used to transform E.coli DH10B cells. Thermo Scientific InsTAclone PCR Cloning Kit (#K1213), the final extension step may be prolonged to 30 min to ensure the highest efficiency of 3'-dA tailing of the PCR product. Clone the gBlock ordered into pJET1.2/blunt vector using CloneJET PCR Cloning Kit (Thermo Scientific) using the manufacturer protocol to generate the entry vector. (GeneAll, Seoul, South Korea), cloned into the CloneJET PCR vector (CloneJET . Cloning and sequencing of RT-PCR products . Ligate the PCR product into pJET1.2 following instructions of CloneJet PCR cloning kit (Figure 2A). 84. For PCR reaction, use a high fidelity polymerase. Remove non-ligated 3DA using RNAClean XP Kit. NEB PCR Cloning Kit. As a result, only bacterial cells with recombinant plasmids are able to form colonies.