LAB2 - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

EGB2420240301092228756.jpeg

material#LAB2

Summary:

• Gel#1 on plasmid DNA.
• Plasmid DNA dilution.
• Preparation of a PCR reaction.
• Make liquid YPD medium for LAB3.
• Make solid SD medium for LAB4.

1. Unfreeze the plasmids from LAB1
2. Vortex the tube to mix the content. If necessary, spin the tube for ~2-3 seconds to collect the liquid at the bottom.
3. Take out 10 µL of the content to a fresh tube.
4. Add 2 µL 6 x loading buffer to your plasmid DNA.
5. Put the gel in the gel tray in a square petri dish. The gel should have at least one well per group number and one extra for the marker.
6. Add 5 µL of the plasmid DNA to an empty well, start with the leftmost well.
7. All group members should load their plasmid.
8. Take note of where your samples are.
9. The teacher will help you to put the gel in the electrophoresis chamber.
10. The teacher will load the molecular weight marker.
11. Apply the electrical field as soon as you are done.
12. The electrophoresis last around ⌛15 min at ⚡200 volts in the Bachman gel tank using a homemade rectifier.
13. When the gel run is completed, the teacher with take a picture using a transilluminator.

• Put the gel in TAE + Midori Green, incubate 15-30 min
• Take picture

1. Pipette 1 mL (1000 µL) ultra-pure water into a clean 1.5 mL Eppendorf tube
2. Mark this tube well so that you can identify it.
3. Transfer 5 µL of the plasmid DNA to the tube with 1 mL water This will make a 5 µL/1000 µL = x 200 dilution.
4. Vortex the tube with the x 200 plasmid dilution so that the content is well mixed.
5. Put the tube with concentrated plasmid DNA back into the freezer.
6. Leave the tube with your diluted plasmid DNA on the bench and continue with the next step.

Each student should prepare one PCR reaction. Add the reagents in the order given below in a 200 µL PCR tube. Pipette very carefully, there is only a limited amount of reagent which is expensive.

PCR amplification (50 µL):

• 33 µL 1.66 x PCR mastermix (= 2x PCR mastermix with 6x loading buffer , hhis is a green solution that has both PCR master mix and loading buffer)
• 5 µL primer 1 (5 µM, check the google sheet for your PCR reaction)
• 5 µL primer 2 "
• 7 µL of the x 200 diluted plasmid DNA.

Table#4	Primer1	Primer2	Template	PCR target	Expected size (bp)
	1113	987	pBR322	amp	1072
	1196	1195	pBR322	pBR	1497
	984	983	YEplac195	2µ	1644
	1804	1347	YIplac204	TRP1	942
	978	977	pYPKpw	Δcrp	445

See see pTAx assembly strategy

The teacher will take the tubes to the BioRad T100 thermal cycler in the LGM laboratory.

Each group should make 100 mL liquid YPD medium in a 250 mL Schott flask. Put a small piece of autoclave tape on the lid. Label the flask properly (Content, Date, Course, Turno, Group etc.).

colored.png