Worksheet 3_ Attempt review La Trobe University BIOMEDICAL MED3LAB
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Course
MED 3LAB BIOCHEM & MEDICAL BIOLOGY
Institution
MED 3LAB BIOCHEM & MEDICAL BIOLOGY
Worksheet 3: Attempt review
My LMS Subjects / 2021-PSB-MED3LAB(SP-T3-PT) / Assessment / Worksheet 3
Started on Tuesday, 12 October 2021, 10:45 PM
State Finished
Completed on Wednesday, 13 October 2021, 11:27 PM
Time taken 1 day
Marks 19.50/19.50
Grade 24.50 out of 24.50 (100%)
Information
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worksheet 3 attempt review la trobe university biomedical med3lab
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11/29/21, 2:01 AM Worksheet 3: Attempt review
My LMS Subjects / 2021-PSB-MED3LAB(SP-T3-PT) / Assessment / Worksheet 3
Started on
Tuesday, 12 October 2021, 10:45 PM
State
Finished
Completed on
Wednesday, 13 October 2021, 11:27 PM
Time taken
1 day
Marks
19.50/19.50
Grade
24.50 out of 24.50 (100%)
Information
Laboratory Module 3 Assessment
In this laboratory module, you prepared growth media and agar plates. You also prepared competent E. coli
cells for plasmid transformation using the heat shock method. In working through the 'dry module', you will
have considered the importance of the different ligation reactions that were prepared in practical 2. In
addition to transforming the E. coli cells with the pQE30-GFP construct (the “vector plus insert” test ligation
sample) and the “vector only” and “insert only” ligation controls, you will also set up a control to check on
the viability and integrity of the competent cells. Transformed cells will be selected for by plating them out
onto agar plates containing ampicillin antibiotic selection. It is possible to determine the transformation
efficiency of the E. coli XL1-Blue cells you prepared as part of this practical.
A part of this assessment asks that you re-visit some some of the important features and consideration in
using the pQE30 vector. You will also need to examine different sets of plates that have resulted from the five
transformation experiments. You will be able to provide possible reasons that could account for the
differences in the number OR type of colonies observed following transformation.
In Part A of Laboratory module 3, you were asked to consider the preparation of agar plates for use in the
transformation procedure.
Refer back to the laboratory manual and consider the steps in the protocol to answer the questions below.
1. Melt your LB agar in the microwave and be very careful not to let it over boil. Patiently heat it up and stop
frequently to swirl the flask.
2. Allow the agar to cool to about 60°C (just cool enough to handle but no cooler).
3. Add sufficient 25 mg/ml ampicillin stock to give a final working concentration of 100 μg/ml. Swirl the flask to
mix the ampicillin throughout the solution.
4. Also, add sufficient 100 mM IPTG stock to give a final working concentration of 0.1 mM. Again, swirl the flask to
mix the IPTG throughout the solution.
What volume of the ampicillin stock is required at STEP 3?
600
µl
What volume of the IPTG stock is required at STEP 4?
150
µl
Ampicillin
Stock concentration = 25 mg/ml (C1)
Final concentration = 100 μg/ml = 0.1 mg/ml (C2)
Total volume of LB agar = 150 ml (V2)
Volume required (V1) = C2/C1 x V2 = 0.1/25 x 150 ml = 0.6 ml = 600 μl
IPTG
Stock concentration = 100 mM (C1)
Final concentration = 0.1 mM (C2)
Total volume of LB agar = 150 ml (V2)
Volume required (V1) = C2/C1 x V2 = 0.1/100 x 150 ml = 0.15 ml = 150 μl
When competent cells are made within a laboratory, they should be evaluated for transformation efficiency. By
calculating the number of colonies obtained using a standard amount of DNA, you will have a measure of how
competent your cells are. This will allow you to compare different batches of competent cells that are prepared
either at different times or prepared using alternative methods.
In PART C of Laboratory Module 3, a positive pQE30-GFP control plasmid was used to monitor the integrity and
viability of the competent cells. The number of colonies obtained on the plate following transformation and
overnight incubation was 2820.
From the protocol we know that:
10 µl of a 1 ng/µl stock of plasmid was used
200 µl of competent cells were used in the transformation procedure
0.5 ml of LB was added to the cells during the transformation process
200 µl of the cell suspension was plated on the agar plates
What was the transformation efficiency of the E. coli XL1-Blue cells used in the lab?
1 x 10^6
cfu/µg
Note: The answer can either be in full numbers or in scientific notation. For scientific notation, your response should only
be to one decimal place.
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