Bio 101L Week 7 Post Lab Name:

Lab Section Number:

TA Name:

“Scientists are Control Freaks” (Daniele Armaleo)

This post-lab focuses on the controls that were set up along side your experimental
ligation/transformations this week in the lab. We have provided a short description of negative and
positive controls for you as a separate document in this folder (Using Controls). We recommend
you read this before you do this assignment.

The following controls were set up this week in lab along with your experimental ligations. These
controls were set up with the same reagents (ligase buffer, ligase, competent cells etc.) and DNA
as you used for your experimental ligations and according to the same protocol (i.e. 1 μl of ligation
mix was added to 25 μl of competent cells, incubated on ice, heat shocked etc).

Ligation and transformation Controls

Control 1 (No DNA control): Add 2μl of water to the 8μl ligation reaction mix.

Control 2 (pBS Vector DNA alone; No insert DNA): Add 1μl of pBS Vector DNA (20 ng/μl) that
has been treated with Eco R1 and phosphatase and1μl of water to the 8μl ligation reaction mix.

Control 3 (Insert DNA alone; No vector DNA) Add 1μl of Eco R1-cut Yeast genomic DNA
(~40ng/μl) and1μl of water to the 8μl ligation reaction mix.

Transformation control
Control 4 (uncut plasmid control) Add 1μl of pUC plasmid (50 pg/μl) to 25 μl of competent cells
and carry out the transformation according to the procedure.

The table below shows some of the results from one of the experiments done
in lab this week. The total number of colonies is the number of colonies that
grew on the LB agar plates supplemented with Amp, X-gal and IPTG. Cells
filled in as “-----“ do not necessarily have a value of zero; rather, they
represent data we have not given you.

Ligations/ Total number of colonies Number of Number of

transformations (transformants) Blue White
Control 1 ----- ----- -----
Control 2 10 colonies ----- -----
Control 3 0 colonies ------ ------
Control 4 200 colonies ------ ------
Experimental 100 colonies ------ ------

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 Bio 101L Week 7 Post Lab Name:
Lab Section Number:
TA Name:

1. What is the purpose of including control 1? (Include in your answer whether this is a negative or
positive control and how many colonies you expect to recover in control 1). (3 points)

2. For control number 2, ten colonies were recovered.

a) Based on the components of this ligation, in a “perfect” world (where molecular reactions work
100% all of the time), how many colonies would you have expected? Why? (6 points)

b) Give a reasonable explanation as to why colonies grew from this control In your explanation
include, out of the ten colonies recovered for control 2, how many of them would you expect to be blue
and how many would be white? What allows you to know whether these colonies took up a pBS
vector? (Remember Ockam’s razor: http://en.wikipedia.org/wiki/Occam%27s_razor ) (4 points)

3. Explain why control 3 had no colonies. (3 points)

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 Bio 101L Week 7 Post Lab Name:
Lab Section Number:
TA Name:

4. The chart indicates that the experimental ligation yielded 100 transformants. Is there any
information, based on the controls, that would help you estimate what percentage of those 100
colonies would be white or blue? Explain. (3 points)

5. Control 4 was carried out with a plasmid that is provided by New England Biolabs (NEB) with the
competent cells. The company provides this plasmid as a form of “quality control” for the
investigator to test whether the competent cells are capable of being transformed. The plasmid
(pUC18) is a circular plasmid and has all of the same features as pBluescript (i.e. the Lac Z gene,
Amp R gene and an origin of replication). In the specification information that is provided by the
company with the competent cells, NEB defines the transformation efficiency of the competent
cells as the number of colony forming units (cfu) which would be produced by transforming 1 µg of
pUC18 plasmid into a 25 µl volume of competent cells (For our purposes a colony forming unit is
equivalent to a colony). Based on the number of colonies that were recovered from control 4,
determine what the transformation efficiency of the competent cells was. You may assume that all
of the transformation mix was plated on the LB Amp, X-gal, IPTG plates. (3 points)

6. An additional control that you set up was at the very end of the lab when you spread a small
volume of the transformation mix onto an LB plate (without Ampicllin, X-gal or IPTG). Assuming
that: 1) the cell density of the transformation mix was 4 X 109 cells per ml and 2) you spread 25 µl
of the transformation mix onto the LB agar plates, estimate approximately how many “colonies”
you would expect to get on this plate? Would you expect the colonies to be white, blue or a mix?
Show your work and explain your reasoning. (5 points)

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 Bio 101L Week 7 Post Lab Name:
Lab Section Number:
TA Name:

7. When cloning DNA fragments into a plasmid vector, the recommended molar ratio of insert:
vector DNA is around 2:1. Determine the molar ratio of insert to vector DNA in the experimental
ligations you did this week. You may base this calculation on the assumption that the yeast Eco
R1 DNA fragments are 3 kb in length (3 points).

My signature below verifies that I have completed this post lab in accordance with the
Duke Community Standards:____________________________________