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.

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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)

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