Bio-rad Ligation and Transformation Module Manuel d'utilisateur télécharger pdf (Page 45)

Detailed Protocol for Transformation

Preparation of Competent Cells

1. Approximately 20–40 min prior to starting the transformation, prepare

competent cells by pipeting 150 µl of fresh starter culture (inoculated

one day prior) into the prewarmed C-growth medium and placing in a

shaking 37°C water bath or incubator for 20–40 min.

Note: If a shaking waterbath is not available, manually shake the culture

tubes every 5 min during the 20–40 min growth phase to oxygenate the

culture.

Note: Your instructor may have already completed this step to save time.

2. Label 1–2 LB Amp IPTG agar plates with your initials. Also label one

agar plate for your ligation (pJet + your insert name) and if you are

performing a positive control transformation label the second agar plate

for the positive control plasmid.

Place agar plates at 37°C.

3. If not already done, pipet 1.5 ml of C-growth medium to the 15 ml culture

tube. Label tube with your initials and warm it at 37°C for at least 10

min.

Also ensure that the

E. coli

starter culture is at 37°C.

4. Label a microcentrifuge tube with your initials and "competent cells".

5. Prepare the transformation buffer by combining 250 µl of transformation

reagent A and 250 µl of transformation reagent B into a microcentrifuge

tube labeled "TF buffer" and mix thoroughly with a vortex mixer (if

available). Keep on ice until use. (Note: This mixture must be used on

the day of preparation.)

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Bio-rad Ligation and Transformation Module Manuel d'utilisateur Page 45