Teacher Notes for Activity 2 Choosing a Plasmid
Choosing the proper plasmid for your cloning experiment is very important. There are many
options available and being able to choose one that will be optimal for your particular
experiment can be daunting! In this activity, you will examine four commonly used Promega
plasmids to determine their characteristics and which will be the best choice for your
“experiment.”
Examine the four plasmids provided. Your options are pBR322, pCAT® 3-Basic, pF1A T7
Flexi® and pGEM® 3Z and answer the following questions. (Note: the plasmids that are
included in the student handout are also available as a PowerPoint slide (plasmids.ppt) so that
you can project them if you care to discuss them in class. Answers to questions are provided in
bold italics below.
1) Which plasmid(s) will grow on plates treated with Ampicillin?
All of the plasmid have Amp’ resistance
2) Which plasmid(s) will grow on plates treated with Tetracycline?
Only pBR322 has Tet’ resistance
3) If you choose pBR322 as your vector and digest it with Sca l and insert your gene of
interest, will it be able to grow in the presence of Ampicillin? Why or why not?
It will not grow in the presence of Ampicillin because the resistance gene will have been
altered by the insertion of the foreign DNA
4) Which plasmid(s) have a restriction site for Hind lll?
pBR322, pCAT® 3-Basic, and pGEM® 3Z
5) Do any of the plasmids have more than one restriction site for the same enzyme? If so,
would that be a good enzyme to choose for your digests? Why or why not?
Yes, some of the plasmids have multiple restriction sites for the same enzyme (ex.
pCAT® 3-Basic has two sites for Hind lll. This would not be a good choice since we only
want one opening in our plasmid.
Teacher Notes for Activity 2 Choosing a Plasmid
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Teacher Notes for Activity 2 Choosing a Plasmid
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Now that you have examined the plasmids, choose which plasmid you would use considering
the following parameters:
Restriction enzymes can be expensive; therefore, it is often a good idea to use an enzyme that
you or colleagues already have in the lab. The same is true for antibiotics. In your lab you have
Ampicillin and Tetracycline; you also have the restriction enzymes Bam Hl (G'GATCC) and Eco
Rl (G’AATTC). You are going to be inserting a 550 kb fragment of DNA into your chosen vector.
The sequence of your fragment is found at the end of this document or in the separate
document sequence.doc that your teacher can provide you. (Note: You may want to load the
document with the plasmid sequence (sequence.doc) on to the computers students will be using
so that they can easily cut and paste the sequence.) Your sequence must be cut with the same
enzyme as the plasmid. Use the NEB cutter online tool found at the following url:
(http://tools.neb.com/NEBcutter2/index.php) to determine restriction sites within your sequence.
In NEB cutter, you will cut and paste your sequence into the pane, select “all commercially
available enzymes” and click “submit.” The cutter will show you all the restriction enzyme sites
within your sequence.
Which plasmid, restriction enzyme and antibiotic should you choose for your experiment?
WHY?
The NEB cutter data will show that our sequence does not have a restriction site for Bam
Hl. It does have one site for EcoRl. Therefore, we will digest with EcoRl. Since our lab
routinely uses tetracycline, the plasmid of choice will be pBR322. Since the EcoRl site
does not interfere with the Tet’ resistance that will be the best choice for our experiment!
Teacher Notes for Activity 2 Choosing a Plasmid
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Contextual Biology Integrated Projects
Created by the Center for Occupational Research and Development
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Online Sequence
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tgtcttgttt
ggagattagg
gagaaagtta
tcatttgaca
ccatgctgac
acaagccgct
ctcagggtca
ctcacacatg
aggggccttc
ggagacagca
cagggggtag
accagaccct
caacaacaaa
aaaagagggt
gagtaaaggg
gttcatagct
ggagggtctt
ttctttagag
gagaggggag
gttcttgtgt
aaatttttgg
agggaggaag
gacaagtaga
gtcactctgg
agtgcaccat
gctcgtgggc
ctgtgtttag
atcacattat
atatgccttc
gtccagcaac
tgcagaggct
aggcaggtga
aaaattagag
tatgatttgg
aatttttctt
gggcatggta
tggaggccag
agagaggaga
aggagagagg
gttcattggt
Teacher Notes for Activity 2 Choosing a Plasmid
Page 4 of 4
aaacaagtat
taaacaaggg
tgagatggtt
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atatgcatat
acctgtctca
tgtcgctggg
catgtgacta
tctaacatag
cactcctaga
taggagatag
ctgaccttgt
acagatatcc
aatgcactac
ggcaaaaggg
gctcatacgt
gagtttgaga
gaggggagag
gcagaagaga
ggagatgctg
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aggagaggag
atagggacga
aatcaaaaca
gtaacaggga
gctctagcct
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tcagagggta
gaggaaggag
agccaggctt
ctgaatctca
ccaaatatgt
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agaagttcac
gtaatcacag
ccagcctggg
gggagaggag
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catagttgaa
aaaggcagga
atgaagctag
gccacccagg
acagcagccc
cagctcaggc
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gagccgcaca
aggtaataag
ttcaggccag
gaatctgctg
tatgtaaaat
caaatttatt
aggcacgtgc
ataagctgct
ctatgtggga
caatgtgatg
agagaagaca
tctgaaggct
ggtgttcttt
aagaaagtga
agaggagaga
aattgtgaaa
cagtccttgt
cggctggagc
ttcacttagc
agtggcacca
tcctgtctgg
cacagggagg
caaacaaggg
ctacctgact
gcagtgtcaa
caggaataag
agtgagggaa
tagaaacaga
ggctgaggcg
aggctccagt
ggggacgggg
caaagccaga
tgacagcatc
Contextual Biology Integrated Projects
Created by the Center for Occupational Research and Development
http://www.cordonline.net/HiESTbiology