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Topo ta cloning kit invitrogen manual
Coli stain,.g., TOP10, DH5, XL-1Blue.
Our protocol solves this problem by employing the Gateway cloning technique to produce expression clones for Agrobacterium -mediated plant transformation.
Finally, our protocol offers the potential for use in high-throughput applications because many of the reactions can be modified for parallel processing.
Coli and, agrobacterium, respectively.
Coli ccdB gene (note: the ccdB protein interferes with.In case a low yield or low quality plasmid DNA is obtained from Agrobacteria, the plasmid can be transformed into.Agrobacterium -mediated transformation for gene functional studies.In our previous work, we created Entry clones by first generating a PCR product containing att, b sites, which required two overlapping PCR reactions due full game rome total war to the addition of a 25 new patch for cod 5 bp att, b site on both ends of the PCR product.Coli strains,.g., DH5).N: plasmid identity of corresponding lane was Entry clone confirmed by DNA sequencing.The plasmid DNAs isolated from a pool.In contrast, the pentr vector can only replicate.Second, each of the pMDC series binary vectors harbors both.Performing the LR recombination reaction The Gateway LR Clonase enzyme mix kit (Invitrogen) was used to perform the LR recombination reaction:.However, this solution is flawed because it involves the selection of an enzyme that only digests the recombinant by-product plasmid, not the target gene and the binary vector.Note : Since the unreacted Entry clones are removed by the selection due to lack of Agrobacterium replication origin, only the recombinant Destination plasmid can be selected in Agrobacterium.
Add 1 mL of 20 mM ice-cold CaCl2 to resuspend the cells gently on ice.
LB: T-DNA left border.
Agrobacterium -mediated T-DNA transformation.Add the following components to.5 mL microcentrifuge tube and mix: Entry clone (100300 ng) 15 L Destination vector,.g., pMDC (150 ng/L) 1 L 5 LR Clonase Reaction Buffer 2 L TE buffer, pH8.0 to 8.Grow a colony of Agrobacterium in 2 mL YEP liquid media by incubating at 28C overnight with shaking (250 rpm).In general, the Gateway technology involves a two-step process.The positive colonies will grow.E: plasmid identity of corresponding lane was Expression clone confirmed by DNA sequencing.Moreover, our protocol improves upon previous methods by utilizing a more straightforward strategy which eliminates setia hujung nyawa episod 24 the potentially time-consuming step of selecting an appropriate enzyme to digest and remove the unreacted Entry clones from the LR reaction, as previously suggested.This is the Entry clone.
Alternatively, as noted above, the use of a restriction enzyme to digest the undesired plasmid after the LR reaction has been reported.
The schematic diagram of this protocol is presented in Figure.