DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA) and their sequences. All oligomers were purified in-house using denaturing PAGE purification. The templates contained a 5’ phosphate modification in order to circularize the template. Briefly, the template to be circularized was annealed to a 20 mer linker, which was complementary to the first ten and the last ten nucleotides of the linear template. After annealing, we used T4 DNA ligase on the template/linker to connect the 5’-P and 3’-OH, forming a circle. The circle was then purified of its linear product and the linker by running through a PAGE gel and subsequent extraction.
- circular template - 5' Phosphate modification
- linker DNA 2X Circular template :** **complementary to the first ten and the last ten nucleotides of the linear template.
- Add T4 ligase 1 microL for 1 microgram
- 20Hours 20C
- ethanol precipitation
- purify on 10% PAGE gel - poured into a SDS protein gel
Article TitleObserving in-vitro DNA synthesis using beta clamp
Circularize DNA oligos of very small size -
The g-complex loads the b-clamp to the p/t and b-clamp then slides on the DNA bidirectionally. In order to ensure that the b-clamp does not slide off the edge of the p/t one can either block the ends of the DNA using biotin-streptavidin or use circular DNA. In our unpublished experiments, we see significant inhibition of polymerase activity due to the presence of bulky groups like streptavidin, hence we proceeded to use circular template for our work.