What is attB1 and attB2?
ATG. Table 1 provides examples of primer pairs (attB1 and attB2) which can be used to amplify genes for specific protein expression formats. Shine-Dalgarno (E. coli ribosome binding sequence) and Kozak (eukaryotic ribosome recognition sequence) show are typical sequences.
What is attB sequence?
The attB site is a short DNA sequence (less than 30 bp) corresponding to the crossover region at which strand exchange takes place (7).
What is a TOPO vector?
TOPO cloning is a molecular biology technique in which DNA fragments are cloned into specific vectors without the requirement for DNA ligases. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3′-end of the PCR products.
What is attL and attR?
The attL and attR sites are substrates for the excision reaction, and each is composed of one B-type half-site and one P-type half-site.
What is BP and LR reaction?
The BP reaction creates an attL-flanked entry clone. The LR reaction creates an expression clone with all of the components necessary for gene expression. The LR Reaction takes place between the attL sites of the generated entry clone and the attR sites of the destination vector.
What is the function of the CCDB gene in our plasmid?
The ccdB gene, located on the F sex factor plasmid of E. coli, is part of a toxin-antitoxin system encoded by the ccd operon, which is responsible for plasmid maintenance during cell division.
What is the role of the CCDB gene in the donor and destination vectors?
The ccdB gene is present in the donor vectors and the destination vectors prior to recombination, and it is exchanged with the gene of interest during the BP or LR reactions. Since the CcdB protein inhibits the growth of CcdB sensitive E. coli strains, most colonies should contain the desired, recombined construct.
What enzyme is used in TOPO cloning?
enzyme DNA topoisomerase I
The key to TOPO cloning is the enzyme DNA topoisomerase I, which functions both as a restriction enzyme and as a ligase. Its biological role is to cleave and rejoin DNA during replication.
How do I add biotin-attb2-oligo (dT) primer to my kit?
If you will be using the 2.3 kb RNA control supplied with the kit, add 5 µl of DEPC-treated water to 4 µl of the control mRNA for a total volume of 9 µl and a final mRNA amount of 2 µg. To your diluted mRNA (mRNA + DEPC-treated water), add the Biotin-attB2-Oligo (dT) Primer and 10 mM dNTPs according to the following table.
How is attB-flanked cDNA cloned into a vector?
Through site-specific recombination, attB-flanked cDNA is cloned directly into an attP-containing donor vector without the use of restriction digestion or ligation. The resulting Gateway entry cDNA library can be screened with a probe to identify a specific entry clone.
How do you use pdonr 222 plasmid?
pDONR 222 is supplied as 6 µg of supercoiled plasmid, lyophilized in TE buffer, pH 8.0. To use, resuspend pDONR 222 plasmid DNA in 24 µl of sterile water to a final concentration of 250 ng/µl.
How do you mix pdonr 222 and cDNA for BP reactions?
The following protocol uses 30-40 ng of cDNA and 250 ng of pDONR 222 in a 10 µl BP reaction. Use 30 ng of your 2.3 kb RNA control cDNA for the BP reaction. If the attB-flanked cDNA sample is greater than 4 µl, see below for necessary modifications. Add the following components to a sterile 1.5 ml microcentrifuge tube at room temperature and mix.