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 lab
prepare
•
LB agar plates (<1 month before)
2 LB agar plates for streaking to use in part B
16 (gives 4 extra) LB agar plates for part C
12 (gives 4 extra) LB agar plates with ampicillin and kanamycin
with kit—check for 2 LB/amp and 2 LB/kan for control group
•
Aliquot for 6 groups
Part 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
•
ice
set 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/gm
o.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
GAATTC
CTTAAG
Part A
Samples 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