Activity #2: Dry-Lab: Choosing a Plasmid

Activity #2: Dry-Lab: 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 on the following page. Your options are pBR322, pCAT® 3-Basic, pF1A T7 Flexi® and pGEM® 3Z and answer the following questions.

1) Which plasmid(s) will grow on plates treated with Ampicillin?

2) Which plasmid(s) will grow on plates treated with tetracycline?

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?

4) Which plasmid(s) have a restriction site for Hind 111?

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?

Activity 2 Choosing a Plasmid
Page 2 of 6 / Contextual Biology Integrated Projects
Created by the Center for Occupational Research and Development
http://www.cordonline.net/HiESTbiology

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. 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?

Online Sequence

061 tgtcttgttt aaatttttgg aaacaagtat atattacttt catagttgaa aagaaagtga

121 ggagattagg agggaggaag taaacaaggg aggagaggag aaaggcagga agaggagaga

181 gagaaagtta gacaagtaga tgagatggtt atagggacga atgaagctag aattgtgaaa

241 tcatttgaca gtcactctgg ctccctgacg aatcaaaaca gccacccagg cagtccttgt

301 ccatgctgac agtgcaccat atatgcatat gtaacaggga acagcagccc cggctggagc

361 acaagccgct gctcgtgggc acctgtctca gctctagcct cagctcaggc ttcacttagc

421 ctcagggtca ctgtgtttag tgtcgctggg ttatacactg gacaagtacc agtggcacca

481 ctcacacatg atcacattat catgtgacta gcacctacta gagccgcaca tcctgtctgg

541 aggggccttc atatgccttc tctaacatag tcagagggta aggtaataag cacagggagg

601 ggagacagca gtccagcaac cactcctaga gaggaaggag ttcaggccag caaacaaggg

661 cagggggtag tgcagaggct taggagatag agccaggctt gaatctgctg ctacctgact

721 accagaccct aggcaggtga ctgaccttgt ctgaatctca tatgtaaaat gcagtgtcaa

781 caacaacaaa aaaattagag acagatatcc ccaaatatgt caaatttatt caggaataag

841 aaaagagggt tatgatttgg aatgcactac tgtaaaccac aggcacgtgc agtgagggaa

901 gagtaaaggg aatttttctt ggcaaaaggg agaagttcac ataagctgct tagaaacaga

961 gttcatagct gggcatggta gctcatacgt gtaatcacag ctatgtggga ggctgaggcg

1021 ggagggtctt tggaggccag gagtttgaga ccagcctggg caatgtgatg aggctccagt

1081 ttctttagag agagaggaga gaggggagag gggagaggag agagaagaca ggggacgggg

1141 gagaggggag aggagagagg gcagaagaga attccttggt tctgaaggct caaagccaga

1201 gttcttgtgt gttcattggt ggagatgctg ttcctgggca ggtgttcttt tgacagcatc

Activity 2 Choosing a Plasmid
Page 2 of 6 / Contextual Biology Integrated Projects
Created by the Center for Occupational Research and Development
http://www.cordonline.net/HiESTbiology