On comparison of amplified products, the gradient PCR emerged as the amplification method of choice for tphB.
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pSB1C3-mRFP1 (insert contains red chromoprotein) and pET24a-IF3 (bacterial initiation factor 3) vectors were digested separately in r-CutSmart buffer, using high fidelity PstI and EcoRI together with fast AP, to ensure de-phosphorylation of the backbone during digestion.
These were then ligated with tphB (also digested with PstI and EcoRI) and transformed into chemically competent DH5α-cells. The cells were plated and grown overnight at 37ºC (see Figure 4). Note that the red chromoprotein on the cells containing the pSB1C3-mRFP1 vector, indicating religation (should our preventative measures with de-phosphorylation fail) and could thus be easily eliminated as containing our recombinant plasmid of interest.
These were then ligated with tphB (also digested with PstI and EcoRI) and transformed into chemically competent DH5α-cells. The cells were plated and grown overnight at 37ºC (see Figure 4). Note that the red chromoprotein on the cells containing the pSB1C3-mRFP1 vector, indicates religation (should our preventative measures with de-phosphorylation fail) and could thus be easily eliminated as containing our recombinant plasmid of interest.
(see Figure 4).
Note that the red chromoprotein on the cells containing the pSB1C3-mRFP1 vector, indicating religation (should our preventative measures with de-phosphorylation fail) and could thus be easily eliminated as containing our recombinant plasmid of interest.
