Plasmid transformation can be used to: introduce an exogenous DNA molecule into a recipient cell to enable it to acquire a new genetic trait.

Operation method

Direct yeast transformation of plasmids

Principle

The gene to be tested was fused to the DNA-binding domain of Gal4 or LexA or other suitable proteins to construct a bait plasmid; the bait plasmid was transformed into a yeast cell line lacking the promoter of the reporter gene, and the yeast that was transformed was selected; and the library plasmid was then transformed into the yeast

Materials and Instruments

Single colony
PLATE solution Vector DNA Transformation plasmid DNA
Centrifuge Tubes Centrifuge

Move

1、 Pick a single colony (2-3mm in diameter) from the plate with a toothpick and transfer it to a 1.5ml sterile centrifuge tube.

2. Mix 10 μl of vector DNA (100 μg) with 10 μl of transforming plasmid DNA and shake well. (If the transforming DNA is made by the "mini-prep DNA" method without RNAase treatment, no vector DNA is needed to be added.)

3. Add 0.5 ml of PLATE solution and shake.

4. Place on the laboratory bench and incubate at room temperature for 4 days.

5. Heat-excite at 42℃ for 15 minutes.

6、 Centrifuge the cells at 8000~10000r/min for 10 seconds to precipitate the cells, carefully discard the supernatant, gently suspend the cells into 200μl of sterile distilled water, so that the cells are evenly suspended, and then directly apply the mixture to the selection plate.

Caveat

Add reagents in strict order

Common Problems

The main obstacle to transformation in yeast, pulsatilla and plant cells is the cell wall. Digestion of the cell wall with enzymes results in efficient transformation.

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