Ch. 2A: How Do You Begin to Clone a Gene?

Learning goals

Describe the characteristics of plasmids

Explain how plasmids are used in cloning a gene

Describe the function of restriction enzymes

Explain how to use restriction enzymes to create a recombinant plasmid

Key IdeasPlasmids - ideal vectors for genetic engineering

– replicate in the bacteria cell– gene promoter– antibiotic resistance as a selectable marker, – can be transferred into bacteria by conjugation.

Restriction enzymes – key to the creation of a recombinant plasmid. – cut DNA at specific sequences – sticky ends allow strands to join

The Plasmid

pARA-R plasmid

Recombinant plasmid of interest

Bruce Wallace

BamH I

Hind III

pARA-R5,302 bp

PBAD-rfp806 bp

pARA-R construct

sticky endBamH I

sticky endHind III

sticky endBamH I

sticky endHind III

3’

5’

5’

3’

3’

3’

5’

5’5’

5’3’

3’

Bruce WallaceEngineering the Plasmid: ligation of rfp gene into p-ARA

BamH I

Hind III

BamH I

Hind III

Restriction digest of pARA-R

Recombinant plasmid of interest

pARA-R5,302 bp

Biotech Experience

PBAD-rfp806 bp

Restriction analysis of pARA-R

Restriction fragments after digest with Hind III and BamH I

Biotech Experience

806 bp

BamH IHind III

BamH I Hind III

4,496 bp

Learning goals: lab #4

Describe why it is important to verify products created in the genetic engineering process

Predict the relative speed of DNA restriction fragments and plasmids through a gel during gel electrophoresis

Separate and identify DNA restriction fragments and plasmids using gel electrophoresis

Key Ideas

The multistep process that is used to clone a gene results in multiple products– Need to verify that you have the recombinant plasmid

you need.

DNA fragments and plasmids can be separated by gel electrophoresis.

Loading dye helps monitor the progress of the gel electrophoresis procedure.

DNA ladder helps determine the sizes of unknown pieces of DNA

Gel is stained in order to show the location of the DNA fragments and plasmids.

Review questions

1. Why is it important to have sticky ends?

2. What is the purpose of the restriction enzymes?

3. How do you confirm the uptake of the gene into the plasmid?

Clone That Gene activity

1. Cut the plasmid and the human DNA with the appropriate

restriction enzyme 2. Insert the insulin gene into the plasmid DNA 3. Determine which antibiotic you would use to identify bacteria that have taken in the plasmid

Tips

Reagents should be stored in a freezer until you are ready to prepare them for students. Allow to defrost for 15 minutes before using.

The reagents can be aliquoted up to several days before the lab, then store in the freezer/refrigerator

Vortex and spin enzyme mix and 2.5x RB before aliquoting

video

Tips

Calibrate and mark the controller

“Floatie” marked with team number. Each period has a different color.

Bottom of tubes in the water

Low-tech water bath

Multiple pans allow tubes from different classes to stay separated