Lab 21 Goals and Objectives:

1
Lab 21 Goals and Objectives Exercise 59
Bacteriological Examination of Water Confirmed
Test check EMB plate for coliforms EDVOKIT300
Blue/White Cloning of a DNA Fragment Transform
E. coli with your ligation reactions (pg
12-13) Each group will need 0.5-10µl and
100-1000µl micropipettors Tips large and
small 2-1.5ml tubes containing pellets of E.
coli on ice CaCl2 on ice RB (recovery
broth) 2 tubes of glass beads 2 plates
(nutrient agar with Amp, X-gal and
IPTG) HOMEWORK calculate the recipe for PCR
reactions to be set up in the next lab. See the
supplemental handout in the packet page 89!
2
Experiment Overview
Ligation
Transformation
EDVO page 6
3
Vector gene we want to clone
ligase incubate
Two possible products -gene ligated into
vector ? -vector religated without gene ?
Transform into E.coli
4
Plate E.coli on medium containing -Amp
select for transformed cells -Xgal turns blue
when hydrolyzed by ?gal enzyme -IPTG induces
promoter
gene ligated into vector ? -disrupts LacZ
gene, -no ?gal enzyme, -colonies
white vector religated without gene? -has
intact LacZ gene, -produces ?gal enzyme,
-Xgal gets hydrolyzed, -colonies turn blue
5
Edvo pg 12-13
6
C1 X V1 C2 X V2
C1 Concentration of stock solution or reagent X
stock or mM/µM stock indicated
V1 How much of the stock reagent you need this
is what you are solving for! in µl
C2 Concentration of the reagent in the final
solution 1X or mM/µM concentration indicated
V2 Volume of the final solution in µl PCR
reactions are 50µl
C1 X V1 C2 X V2 Solve for V1 V1 (C2 X V2)
C1
V1 µl (final conc. X 50µl) stock conc.