Microsoft word - wssp-lab-2b.doc

Plating an Artemia franciscana cDNA Library
During this project you will be screening a library of cDNA clones from the brine shrimp A.
franciscana. (This will be covered in detail in Chapter 1 of the lecture notes.) Your first
experiment in this lab will be to plate bacteria containing genes from A. franciscana. Each
bacterium will grow up to form a colony. Each colony should contain a plasmid with a random,
but unique, A. franciscana cDNA insert. Later this week you will pick a few colonies and grow
each in liquid culture so that you can isolate the plasmid DNA containing the A. franciscana
sequence.
1. Each group should go to the Reagent Bench to obtain:

4 LB + ampicillin petri plates A glass vial of sterile glass beads A 250 ml orange cap bottle containing LB A microcentrifuge tube of 100 mg/ml Ampicillin A microcentrifuge tube of 2% X-gal solution (in dimethyl formamide) A microcentrifuge tube containing a culture of A. franciscana cDNA library Although each group will be sharing reagents, each person will spreading their own plates of bacteria as described below. 2. Each person should use a black ultra fine marker to label the bottom of two LB +
ampicillin petri plates with name and either a “UN” for undiluted or “1:10 dil” to
indicate the sample was diluted 10-fold. Try to write along the periphery of the plate so
you will be able to see your colonies once they’ve grown up.

3. Spread 40 µl of X-gal solution onto each plate.
Pour approximately 15-20 sterile glass beads onto the agar surface of the LB+amp plate. Then add 40 µl of X-gal solution to each plate and cover the petri plate. (Add the X-gal solution in several very small drops distributed evenly over the plate. This will give you very even spreading of the reagent.) Hold each plate upright and shake it from side to side while occasionally rotating the plate a fraction of a turn. (This technique will spread the X-Gal reagent evenly over the surface of the plate.) Pour the glass beads into the large beaker for used glass beads so that they can be recycled.
4. Each person should prepare a bottle of LB broth with 100 µg/ml ampicillin.
To save time and reduce the amount of pipeting, it is best to make up a working solution of
LB broth containing 100 µg/ml ampicillin, which you will be use to inoculate bacterial
colonies. Therefore, each person should prepare LB + Amp solution by adding an
appropriate volume of 100 mg/ml ampicillin stock to a sterile bottle of 250 ml LB broth.
The ampicillin in the diluted mixture will degrade over time. However, you will be using
this LB+amp mixture often during the next week, so the ampicillin is not going to break
down during such a short time. Be sure to use labeling tape to put your name on your bottle of LB + amp broth and store the bottle above your bench.
5. Each person should label one fresh microfuge tube with a “1:10 dil”. Add 180 µl of
LB+ampicillin to the tube.

6. Gently vortex the microcentrifuge tube containing the of the Artemia cDNA library
culture to suspend the cells. Using a P-20, remove 20 µl of cells from the tube and add
it to the LB+amp in the tube labeled “1:10 dil.”

7. Gently vortex the microcentrifuge tube labeled “1:10 dil” containing the diluted culture
to suspend the cells.

8. Spread 100 µl culture of A. franciscana library onto the “UN” plate.
a. Pour approximately 15-20 fresh sterile glass beads onto the agar surface of the “UN” plate.
b. Pipet 100 µl of A. franciscana cDNA library stock you were given onto the “UN” plate.
c. Hold each plate upright and shake it from side to side while occasionally rotating the plate
a fraction of a turn. (This technique will spread the cells evenly over the surface of the plate.) d. Pour the glass beads into the large beaker for used glass beads so that they can be washed
9. Spread 100 µl of the “1:10 dil” culture of A. franciscana library onto the “1:10 dil”
a. Pour approximately 15-20 fresh sterile glass beads onto the agar surface of the “1:10 dil”
b. Pipet 100 µl of the “1:10 dil” diluted A. franciscana cDNA library stock you made onto
c. Hold each plate upright and shake it from side to side while occasionally rotating the plate.
d. Pour the glass beads into the large beaker for used glass beads so that they can be washed
10. Place you plates upside down in the 37oC air incubator.
When the surface of your plates is dry, place them in the 37oC air incubator and incubate overnight. The instructor will remove your plates tomorrow and place them in your refrigerator for you to use later in the week. Name: ______________________________ School: ______________________
Lab 2 Problem Set:
Q: What would you expect to be the relative difference in the number of colonies on your
“UN” and “1:10 dil” LB+amp plates?
Q: When spreading the cDNA library on the LB+Amp plates, why was it necessary to also
spread the X-gal reagent?
Q: Why is there ampicillin in the plates?
Q: What ratio of white to blue colonies do you expect to find on the plates?

Source: http://wssp.rutgers.edu/StudentScholars/WSSP08/WSSP-files/Lab/WSSP-Lab-2.pdf