PCR II (semi-nested): components, their concentrations, and thermal profile were similar to PCR I, except we used 2 μl of PCR I-product as template, substituted primer GIo by primer DRB1.2 [ 40], increased the annealing temperature to 65°C, and increased the number of cycles to 30.
The molecule was assembled by using raloxifene as a template and substituting an indole ring for the benzothioprine core.
Selective amplification reactions were similar to pre-amplifications, with 3 μl of diluted pre-amplification product used as template and substituting 50 pM of the appropriate FAM--labeled EcoRI + 3/250 pM PstI + 2 selective AFLP primers.
The template DNA was substituted with sterile deionized water in the negative control.
Non-template control (NTC) samples contained the same PCR reagents as above but had 2 µl of water substituted for template.
Non-template control (NTC) samples contained the same reagents as above for each of the 7 distinct PCR reactions but had 2 µl of water substituted for template in each case.
A negative control was set up by substituting the template with double-distilled H2O.
Thus, for CTP selection to occur, the preceding templating base (dG) must substitute the favorite dC, but keep the same distance with the 5′P.
A structural modeling of GmF3G2″Gt using the 3D structure of a grape flavonoid 3- O-glucosyltransferase, VvGT1, as a template indicated that three substituted amino acids (V142, S149, T183 in GmF3G2″Gt-b) were located far from the enzyme active sites.
The results indicate that the organic templates are mainly substituted by the conducting polymer polypyrrole without destroying the previous nanotube structure.
