Cloning Lab
This is your culminating biotechnology project. You will work with your partner to complete a series of labs.
A central problem of cloning is the identification of a desired clone among countless bacteria,
transformed and untransformed. In the simple system we have used so far, we have used antibiotic
resistance to select the desired bacteria. In this lab we will need to select for MORE than this. This is
where the concept of Blue/White Screening comes in. The pUC19 plasmid just as pBLU are specially
designed cloning vectors that make use of the lac operon…aka…B-galactosidase…aka lacZ gene. This gene
codes for an enzyme that breaks down lactose as a food source. When X-gal is added to the growth media
on the plates, this enzyme also breaks X-gal down which creates an artificial blue or indigo color. With
the pUC19 plasmid an additional reagent also must be added to the plates called IPTG that helps to kick
this gene into high gear or else not enough enzyme is produced to turn the colonies blue.
So in short these two concepts are very important to understand:
1. E. Coli that produce B-galactosidase and are fed X-gal will turn blue.
2. E. Coli that do not produce B-galactosidase will remain white even in the presence of X-gal.
YOUR TASK: Successfully insert at least one fragment of Lambda DNA into the pUC19 vector, and verify
the size(s) of the fragment(s) by gel electrophoresis.
TIMING:
Here is a rough outline of what labs will need to be completed in this lab. The timing is up to you, with
the exception of the transformation. This is because the transformation will be done with commercially
prepared competent cells that are engineered to really boost your transformation efficiencies. These
cells are very expensive and arrive on dry ice. They remain viable for about 2 days, so your
transformation will need to be done during that timeframe. For this year these cells will be available
starting 5/6, so you should do your transformation no later than 5/7. Below is an outline for you and
your partner to determine when you will perform the labs involved in this project.
LAB
Restriction Digest #1
Pour Gel
Run Gel
Ligation
Transformation
DATE
Inoculation
Plasmid “prep” Isolation
Restriction Digest #2
Pour Gel
Run Gel
Analysis of Results
LAB REPORT DUE AT START OF CLASS
5/18
QUESTIONS TO CONSIDER:
1. What is the procedure to “cut” lambda? Which restriction enzyme do you think you should use?
2. What would be the procedure to cut a plasmid? Which restriction enzyme do you think you
should use on pUC19?
3. Where would the best place be to insert a lambda fragment into pUC19 in order to utilize the
double screening of Ampicillin Resistance AND Blue/White colonies? Why?
4. What color colonies will contain your “clone” meaning the pUC19 with a lambda insert?
5. What procedure must you perform to “glue” your insert (lambda fragment) into your pUC19?
6. Why is verifying the restriction digest success with gel electrophoresis a good idea?
7. What are you doing when you perform a plasmid prep in general?
WHAT TO TURN IN:
Please include these headings in your TYPED report.
1. Background Information
2. Research Question: Which fragment is ligated into my pUC19 vector?
3. Summary of ALL PROTOCOLS broken down into individual labs that correspond to your
timeline.
4. Data:
 Pictures of ALL GELS titled and labeled with band sizes
 Pictures of Plates
 Transformation efficiency calculation
5. Analysis: Which fragment or fragments do you think are ligated into your pUC19? Why? What
evidence do you have?
6. Conclusion and Evaluation: Summarize and justify using your evidence. Deduction of errors
and limitations with evaluation of their effect on your data.
PLEASE NOTE: Your conclusion will be submitted as the C&E portion of an IA to IB. You should
follow the standard IA scoring guide for your conclusion and evaluation.