This preview shows page 1. Sign up to view the full content.
Unformatted text preview: lls. 9) Using the pipetting technique described above, add 5 drops of sterile media (L broth) to both the
"+" and "-" tubes and place at 37oC for 30 minutes.
This is called the "out-growth" step and allows bacterial cells to metabolically recover. This period
also allows time for the replication of the plasmid DNA and the transcription and translation of the
antibiotic-resistance gene mRNA before challenging the cells with antibiotics.
10) During the "out-growth" period we will finish the dilution experiment started in Step 7:
a) Using a new sterile pipette and the technique described in step #2 above, add 3 drops of the
remaining unused E. coli cells to saline tube "A" and mix gently.
b) Transfer 1 ml of the cell suspension from tube "A" to tube "B" and mix gently.
c) Transfer 1 ml of the cell suspension from tube "B" to tube "C"; mix gently.
11) Label the bottom of three petri plates without antibiotics (no colored stripe) with "A", "B" and "C".
Also, put the date and your group name on the bottom of the plate. This ensures that if the tops fall
off (which they do occasionally), you will be able to recover your data.
12) Plate 3 drops of the cell suspensions "A", "B" and "C' on the appropriate plates. Your TA will
demonstrate the plating procedure. Invert the plates and incubate overnight at 37oC.
13) We now return to the transformation experiment (Step 9). Obtain two ampicillin plates (AMP;
blue stripe label) and two nutrient agar plates (LB; no stripe label).
a) Label the bottom of one ampicillin plate with “+AMP”, the date, and your group name. Label
the bottom of one LB plate with “+LB”, the date, and your group name.
b) After the 30-min out-growth period, plate 3 drops of your "+" tube on the +AMP plate and
another 3 drops on an + LB plate.
c) Label the bottom of the second ampicillin plate with “-AMP”, the date, and your group name.
Label the bottom of the second LB plate with “-LB”, the date, and your group name.
d) Plate 3 drops of your "-" tube on the –AMP plate and another 3 drops on the – LB plate.
e) Invert all plates, tape all of the plates together, and incubate overnight at 37oC. Your TA will
oversee the length of the incubation period.
SUMMARY: Each group should be incubating seven plates (total); four from the transformation
experiment (+AMP, -AMP. + LB, and - LB) and three from the dilution experiment (A, B, and C).
14) After 16-20 hr at 37oC, each single growing bacterial cell will have divided thousands of times to
give rise to a mound of bacterial cells which is called a bacterial colony.
Your TA will show you a plate with E. coli colonies and will demonstrate a quick and relatively
easy way to count them.
15) Counting will be done once the incubation is complete – you must arrange a time to count your plates. This will not take more than 30 min of your time. DO NOT disturb any laboratory in
progress. This can be done any time 16 hr after your laboratory period. Arrange for a mutually
convenient time with your TA and your lab partner. The TA has the right to ask for colonycounters to leave and return at another time. Biology 05LA – Fall Quarter 2012 Lab 7 – Page 4 Carefully count and record the number of colonies on each plate. You should be able to provide answers to
the following questions from your data.
a. How many cells were available to be transformed in the "+" and "-" tubes? *
b. What is the percent cells actually transformed? See the worksheet at end of the lab exercise for
help here. SPECIAL EXERCISE
In each laboratory, two groups will undertake a modification of this experiment. These groups
will be given the plasmid pBluescript which, like pUC118, is commercially available. The actual
plasmid sample that will be provided will be a mix of two forms of this plasmid; one will be
pBluescript while the second will be pBluescript into which a small piece of DNA has been cloned
pBluescript, like pUC118, contains an origin of replication, a gene encoding resistance to
ampicillin, and the gene for one subunit of the lacZ gene (coding for the enzyme -galactosidase). galactosidase is comprised of 2 subunits (therefore it has a quaternary structure). This portion of the
lacZ gene also contains a multiple cloning site (MCS) into which extra DNA can be cloned. When pBluescript, lacking extra cloned DNA, is introduced into a host strain of E. coli, the gene
carried by the plasmid can only produce only one portion of the enzyme -galactosidase and therefore
cannot, on its own, cleave this sugar. However, if the bacterial cell genome contains the gene for
encoding the other subunit of -galactosidase, a complete gene for the enzyme is available for
transcription and translation of a fully functional enzyme. This raises the question as to how can we
know that a complete, functional -galactosidase is present within the bacterial cell? T...
View Full Document
This note was uploaded on 08/27/2013 for the course BIO BIOL05LA taught by Professor Abbottl during the Fall '12 term at UC Riverside.
- Fall '12