Week 4

Question 1

Why do you add EDTA in step 2 in practical 3?

Answer 1

To stop DNA polymerase from working, stopping the reaction
If you add a high concentration of EDTA it will stop DNA polymerase from working, thus halting the reaction

Question 2

What reagent will be used to resuspend your purified sequencing products (DNA) before these are loaded into the ABI sequencer?

Answer 2

formamide
vacuum dried and resuspended in formamide as it it maintains the quality and conductivity of the solvent in capillary electrophoresis

Question 3

On the sequencer, the smallest fragments will travel fastest and as such will be the first to move past the detector. Which direction will the resulting sequence be in?

Answer 3

5’ (smallest fragments) to 3’ (largest fragments)
The fragments are synthesised in the 5’ to 3’ direction, so the smallest fragments will be those at the 5’ end.