Difference between revisions of "Part:BBa K1636000"

Revision as of 01:44, 19 September 2015

Lead-II-ions-specific aptamer

This aptameric sequence is capable of recognising and binding specifically to lead II ions. This sequence, which works as a single stranded nucleic acid molecule, is capable of forming a G-quadruplex by interacting with lead ions (Pb2+) while disrupting a partially complementary duplex.

Sequence and Features

The linker was ligated into the linearised plasmid pSB1C3 and previously cut with EcoRI and PstI, then a purification was performed (figure 1A). Several clones were selected to isolate plasmid by miniprep (figure 1). In order to analyse the integrity of plasmids an electrophoresis was run (figure 1B). Several plasmid isoforms were observed and the plasmids were suitable to be used as template in a PCR reaction to know the presence of the insert.

Figure 3. A) Scheme of aptamer cloning in pSB1C3 plasmid and B) electrophoresis with agarose gel at 0.8% to analyse plasmid extraction from different clones (lanes 2 and 3 with 10 microliters of each sample + 4 microliters of loading buffer).

To know if the aptamer was cloned in pSB1C3 a PCR was performed using universal primers VF2 and VR (figure 4A). Those primers amplify a PCR product of 299 pb when the insert is present (figure 3A). Plasmids from 2 different clones were tested and one of them had the PCR product of approximately 299 bp corresponding to the plasmid with insert (figure 4B, lane 3).

Figure 2. Analysis of the aptamer cloning into pSB1C3 plasmid by PCR. A) Plasmid scheme with the position of the cloned aptamer wich is flanked by the restriction sites EcoRI and PstI, also the alignment position of primers VF2 and VR is indicated. B) Plasmids from 2 different clones were used as templates for PCR with universal primers VF2 and V (lanes 2-3). Lane 1 contains Quick-Load® 2-Log DNA Ladder(0.1-10.0 kb), NEB.