The below gel photos showed the result of optimization of DNA duplex performance using 8% DNA Polyacrylamide Gel Electrophoresis (PAGE).
From the result, it is clearly shown that bands appeared in each DNA duplex. Although multiple bands are shown in the lane containing DNA duplex for oligo 9+10, the band at the bottom below the corresponding DNA ladder is expected to be the DNA duplex product. It is suggested that the band intensity in the lane containing oligo 1+2, are the highest compared to lanes containing oligo 3+4, oligo 5+6, oligo 7+8 and oligo 9+10 respectively. It is suggested that the mixture of oligo 1 and oligo 2 possess the highest stability, compared to different DNA duplex, which is selected to be used in the enzyme conjugation.
No band was observed for oligo 5 and oligo 6 in 1st gel and oligo 9 and oligo 10 in 2nd gel. The relative high concentration of the DNA ladder resulted in a rather short exposure time for the gel. These bands are expected to appear when higher concentration of samples is prepared.
Conjugation of DNA oligo and enzyme Conjugation was successful using 50uM of DBCO, with MnP to oligo ratio at 2:1. With the oligo dye, the lower band here is expected to be the oligos without conjugation with MnP, while this band is expected to be the oligos with conjugation. No bands were found even 50uM DBCO used. What we learn is that it may be due to wrong filter size used in purification. The size of LiP is about 47kDa, while the filter size is 50kDa.
