Overview of Transformation Protocol -Prepare competent bacteria for transformation: Treat starter E. coli bacteria with CaCl2and Competent Cell Solution (CCS). Store on ice until transformation procedure. Competent cells are cells that are likely to take up foreign DNA and be transformed. This step increases the likelihood that the E. coli cells will take up the introduced vector and be transformed. -Transformation procedure: Obtain two microcentrifuge tubes containing your competent cells. Label one tube +DNA and one -DNA. Add CaCl2 to both tubes. Add the transformation mix containing the plasmid DNA to the tube labeled +DNA. Do not add any plasmid DNA to the -DNA tube. Incubate both tubes on ice for 10 minutes. Then, place both tubes in a 42\deg C water bath for 45 seconds. Replace the tubes in an ice bucket for 2 minutes. Add recovery broth to both tubes. Incubate both tubes in a 37 C water bath for 5 minutes. Questions: 1) What differences would you expect to see between the -DNA/+Amp and +DNA/+Amp plates? 2) Predict the growth you would expect to see on each of the following plates: – DNA ___________ – DNA/+Amp +DNA/+Amp +DNA/+Amp/+IPTG _ 3) Sketch the results you see from the Rainbow Transformation demo (you will need to draw all four plates. In the space next to the plates, note the following: how much bacterial growth do you observe? How many colonies are present? What color(s) are the bacteria? If there is no growth, why do you think that is?

Human Anatomy & Physiology (11th Edition)

11th Edition

ISBN:9780134580999

Author:Elaine N. Marieb, Katja N. Hoehn

Publisher:Elaine N. Marieb, Katja N. Hoehn

Chapter1: The Human Body: An Orientation

Section: Chapter Questions

Problem 1RQ: The correct sequence of levels forming the structural hierarchy is A. (a) organ, organ system,...

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Overview of Transformation Protocol

-Prepare competent bacteria for transformation:

Treat starter E. coli bacteria with CaCl2and Competent Cell Solution (CCS). Store on ice until transformation procedure.

Competent cells are cells that are likely to take up foreign DNA and be transformed. This step increases the likelihood that the E. coli cells will take up the introduced vector and be transformed.

-Transformation procedure:

Obtain two microcentrifuge tubes containing your competent cells. Label one tube +DNA and one -DNA.

Add CaCl2 to both tubes.

Add the transformation mix containing the plasmid DNA to the tube labeled +DNA. Do not add any plasmid DNA to the -DNA tube.

Incubate both tubes on ice for 10 minutes. Then, place both tubes in a 42\deg C water bath for 45 seconds. Replace the tubes in an ice bucket for 2 minutes.

Add recovery broth to both tubes.

Incubate both tubes in a 37 C water bath for 5 minutes.

Questions:

1) What differences would you expect to see between the -DNA/+Amp and +DNA/+Amp plates?

2) Predict the growth you would expect to see on each of the following plates:

– DNA ___________________________________________________________

– DNA/+Amp ______________________________________________________

+DNA/+Amp ______________________________________________________

+DNA/+Amp/+IPTG _________________________________________________

3) Sketch the results you see from the Rainbow Transformation demo (you will need to draw all four plates. In the space next to the plates, note the following: how much bacterial growth do you observe? How many colonies are present? What color(s) are the bacteria? If there is no growth, why do you think that is?

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