Cloning Cytochrome b PCR Fragment into pGEM-T - Cornell College

Mix well. Add water last and mix the components well by pipetting the reaction up anddown several times with the same tip. Simply tapping the tube is not sufficient to mix.**One of the perennial problems of DNA sequencing occurs when analyzing DNA segments thatare G-rich. Basically, DNA structure dictated by Watson-Crick base pairing is disrupted in G-rich segments because of their ability to create inter and intra strand hydrogen bonding. Thisaggregation causes enzymatic disruption so sequencing and PCR experiments become highlyproblematical. The problem arises from extra hydrogen bonding forming at the N7 position ofG. Traditionally, 7-deaza-G has been used to overcome these problems with some successbecause it eliminates intra strand hydrogen bonding.7-deaza dGTPdGTP3. Label a set of four 0.2 mL tubes A, T, G and C for each template/primer combination.4. Add 4.0 µl of the A Termination mix (purple-capped tube) to the A tube(s), and the TTermination mix to the T tube(s). Do the same for the G and T tubes.5. Add 4.0 µl of the appropriate template/primer combination (master mix from step 2) to eachA, T, G, and C tube and mix well.6. Carefully seal the tubes, place in thermal cycler and start the program.7. Pour a 5.5% acrylamide sequencing gel (8M urea).8. At the completion of the cycling program, add 3 µl of Stop Solution to each tube.9. Denature samples at 92°C for 3 minutes and place on ice prior to loading.10. Pre-run the gel in 0.8X TBE and load the samples (between 0.5 l, and 1.0 l) into the gel.The gel runs for 9 hours.7