LAB6 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

material#LAB6

Summary:

• NaOH total yeast DNA preparation
• Colony PCR

Each student should have a plate with colonies. Do not contaminate this plate, we will need these yeast cells later.

1. Watch this YouTube video (2 min) describing the technique.
2. Add 20 µL 20 mM NaOH to a clean 1.5 mL Eppendorf tube.
3. Pick a small amount of cells from your plate (from LAB5) with a yellow tip. It is important not to take too much and no agarose (see at 52 s in the video).
4. Add the cells to the solution and swirl to mix (see at 59 s in the video).
5. Incubate tubes at 95°C for ten minutes.
6. When the 95°C incubation is over, add 180 µL TE buffer.
7. Vortex the tube for 30 s.
8. Spin at max speed in a micro-centrifuge for 10-20 s.

1. Add 15 µL of PCR mix^* to a new PCR tube (these are the small tubes).
2. Add 5 µL of the yeast DNA to the PCR tube, do not disturb the cell debris from the bottom of the tube.
3. Put tubes in PCR machine (or freeze the tubes at -20°C for later).
4. Run this PCR program:

Taq DNA pol
|95°C  |95°C               |     |
|______|_____          72°C|72°C |
|10min |30s  \ 53.8°C _____|_____|
|      |      \______/ 0:30|5 min|
|      |          30s      |     |

^* PCR mix for 25 reactions (15 µL for a 20 µL PCR reaction): 5. 25 * 13 = 260 µL 1.5x Green PCR mastermix 6. 25 * 1 µL = 25 µL 1222 (10 µM) 7. 25 * 1 µL = 25 µL 1779 (10 µM)