I am a Scientific lab tech at a high school conducting a number of Biotech experiments. As part of the yr 12 Biology genetics and Biotech unit, we run a DNA digest over a couple of lessons. The results aren't bad but can be variable, and I would like some advice on how to improve the results.
Protocol as follows:
λ bacteriophage DNA: ~1ug (3ul)
Buffer : 2ul (10x buffer)
Enzymes : 2 units (2ul) (This is using a dilution of the 12 unit/ul stock supplied from the biotech company into a 1 unit/ul aliquot, which is easier for the students to pipette. The dilution is only made just before each experiment
Water : 13ul (autoclaved RO/DI water, aliquoted in sterile conditions)
- Incubated at 37˚C for 1 hour. I then add 2ul 10X loading dye to stop the reaction, then immediately freeze.
The next class, all tubes (& pre-cut lambda with EcoRI + HindIII) are placed into the water bath at 65˚C for 2 minutes to 'unstick'. Gel is 0.9% agarose with Redsafe dye. 10ul per lane (they make 2 lanes for each tube). Gel is run for 35 minutes at 100V. Gels are then placed on a UV illuminator to take photos.
Lane 1- Control (no enzyme)
5- MWM (pre-cut Lambda with EcoRI + HindIII) 6- 1kb DNA ladder
These are some average photos, some are worse or better. Obviously common students mistakes, poor pipetting, or just forgetting components affect the results. Is there anything else that can be done to help improve these.
Edited by SteveB, 08 June 2022 - 08:26 PM.