Easy Gene Splicer

DNA Ligation and Colony Transformation

Carolina Kit

Timeline

Week Before: read lab & Flowchart, only 6 groups

Monday—Lecture, part A

Tuesday—part B

Thursday--part C

Friday FIRE—come view results

Prep. For labprepare • LB agar plates (<1 month before)

2 LB agar plates for streaking to use in part B16 (gives 4 extra) LB agar plates for part C

12 (gives 4 extra) LB agar plates with ampicillin and kanamycinwith kit—check for 2 LB/amp and 2 LB/kan for control group

• Aliquot for 6 groupsPart A--20uL Ligation Buffer/ATP/ligase (6)--10uL pAMP (5 tubes—pKAN control does not get one)--1ouL pKAN (5 tubes—pAMP control does not get one)Part B--500uL Calcium Chloride (6)--500uL LB (6)

set up for part A • iceset up for part B • Streak starter plates (12-20 hours before part B) (start after school)• 42C water bath• 37C incubator• put calcium chloride on ice• When finish store at 0C (breaker of ice in fridge)set up for part C• 37C incubator

Controls

2 will be a control group:

1. --pAMP+ligase (use 10uL water)

2. --pKAN + ligase (use 10uL water)

--follow same directions, but run these instead of pAMP/KAN

--only plate on LB/amp and LB/kan plate, do not spread on LB plates

Group Assignments

Group 1—pAMP control

Group 2—pKAN control

Group 3

Group 4

Group 5

Group 6

Write-up

• Flowchart (10 points)

• Results and Discussion: Part A and Part B (65 points)

Staple these together. Drawings should be neat!

Review Background Information

• Use website http://bioinformatics.dnalc.org/gmo/animation/gmo.html

• Animations

How did these samples get cut?

Cutting and pasting A & B

Why can restriction enzymes be used for?

Transferring and storing A & B

Important for this lab:• Endonucleases – enzymes that cut RNA or DNA at specific sites; restriction enzymes

are endonucleases that cut DNA• Sticky cells – restriction fragments in which one end of the double stranded DNA is

longer than the other; necessary for the formation of recombinant DNA• Restriction enzyme mapping – determining the order of restriction sites of enzymes

in relation to each other

Restriction enzymes are used for transformation (we will do this soon):• Transformation – the uptake and expression of foreign DNA by a cell• Transduction – the use of viruses to transform or genetically engineer cells• Competent/competency – the ability of cells to take up DNA• Selection – the process of screening potential clones for the expression of a particular

gene, for example, the expression of a resistance gene (such as resistance to ampicillin) in transformed cells

• Transformation efficiency – a measure of how well cells are transformed to a new phenotype

• Recovery period – the period following transformation where cells are given nutrients and allowed to repair their membranes and express the “selection gene(s)”

• Beta-galactosidase gene – a gene that produces beta-galactosidase, an enzyme that converts the carbohydrate X-gal into a blue product

• Green fluorescent protein – a protein found in certain species of jellyfish that glows green when excited by certain wavelengths of light (fluorescence)

• Scale-up – the process of increasing the size or volume of the production of a particular product

Restriction Enzyme Info. (page 216-218)

• rDNA (recombinant DNA)—the produced piece of DNA from inserted another piece of DNA

• recognize specific sites to cut the DNA• Blunt ends—straight across• Sticky ends—one side of DNA is longer than the other, these

overhangs allow for complementary matches between two DNA pieces cut by the same enzyme, the sticky ends match and pasting ma occur to produce an rDNA molecule

• More than 1200 restriction enzymes discovered & isolated from bacteria

• Read 5 3• Palindromic (example radar or

GAATTCCTTAAG

Part ASamples of the plasmid fragments are mixed with DNA ligase and incubated at room temperature for 2-24 hours. Complementary BamHI and HindIII “sticky ends hydrogen-bond to align restriction fragments. Ligase catalyzes the formation of phosphodiester bonds that covalently link the DNA fragments to form stable recombinant DNA molecules.

• Use pipets• After 2-24hours, will store in fridge at 4C

Part B

Transform E.coli with the ligated plasmid DNA. E. coli cells are scraped off an LB agar plate and suspended in two tubes containing solution of calcium chloride. The ligated pAMP/KAN plasmid is added to one cell suspension, and both tubes are incubated at 0C, then heat shocked at 42C, cooling and addition of LB broth, cells then recover.

• I will store cells at 0C (beaker of ice in fridge) after 5-6 hours of incubation

Part C• Samples of the cells are plated on two

types of growth media: plain LB agar and LB agar with ampicillin and kanamycin. Incubate to grow cells. Only cells with both ampicillin and kanaycin will be expressed on the 2nd plate

• Come in at FIRE to see• You are receiving 2 LB plates—so you can

do both plates in PART C step 1.C and 2• Use spreaders at Part C 3