Experiment 2: Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector

Bacteria frequently contain extrachromosomal plasmids, which are circular DNA genetic elements that are self-replicating. Plasmids are typically not necessary for the bacteria's survival but can sometimes confer a growth advantage to the bacteria. Plasmids can be transferred between bacteria and recombinant DNA technology makes use of this feature to introduce “foreign” genes into bacteria and use the bacteria as a cell “factory” to produce the gene. Genes are inserted into plasmids (also called vectors) by means of restriction enzymes. Restriction enzymes are bacterially derived enzymes that recognize specific DNA sequences and cut (or restrict) that particular DNA sequence in a consistent way. That is, any DNA that contains a particular restriction enzyme sequence will be cut the same. Restriction enzymes often produce overhanging ends of DNA (called sticky ends) that can adhere to each other through hydrogen bonding then DNA ligase permanently links the pieces together by forming covalent bonds between the phosphate backbones of the DNA, creating a recombinant or genetically engineered DNA. In this experiment, you will be given two sequences of DNA: one sequence is a foreign gene and the other sequence is of a plasmid vector. You will use a computer program to identify where the common restriction sites for both sequences occur. Scientists perform this type of computer simulation prior to performing restriction enzyme digestion to ensure that they will cut the sequences as expected.

Procedure

1. Type the link listed in the materials box for the NEB Cutter website into a web browser (note, cutting and pasting the link from a PDF file format will not work. You must manually type in the address).
2. Copy and paste the foreign DNA sequence into the box on the NEB Cutter website where it says “Paste In Your DNA Sequence”. Foreign DNA Sequence:
   

   GAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

   CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC

   GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG

   CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGC

   AGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG

   CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCA

   ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA

   TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC

   TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC

   GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC

   TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCC

   CGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC

   GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG

   GACGAGCTGTACAAGGGATCC

3. Title the sequence as “Foreign DNA” in the “Name of Sequence” box. Leave the other settings on their default status.
4. Click the “Submit” button (located to the right of the pasted DNA sequence).
5. On the new page that opens, select “Custom Digest” under the “Main Options” heading on the left side of the page.
6. The new page that opens will contain a list of restriction enzymes. Select the enzymes BamHI and EcoRI then click on the “Digest” button at the bottom of the page.
7. On the new page that opens you will see a linear representation of your DNA. Select “Fragments” under the “List” heading on the right side of the page.
8. Record the length of the longest fragment in Table 1. This fragment contains the sequence of the foreign DNA. The short fragments are the left over ends from the restriction enzyme digest. Close this window.
9. Return to the NEB Cutter homepage. Copy and paste the plasmid gene sequence into the box on the NEB Cutter website where it says “Paste In Your DNA Sequence”. Plasmid DNA Sequence:
   

   GTTAACTACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG

   TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA

   AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCG

   CCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACG

   CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATC

   GAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT

   TCTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTG

   TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT

   TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG

   AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT

   CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG

   GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA

   ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAAC

   TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATG

   GAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGG

   TTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGC

   ACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT

   CAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT

   TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTACCCC

   GGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATT

   GTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTT

   AACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATA

   GGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCC

   AACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATC

   ACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA

   AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCGAACGTGGCGAGAAAGGAAG

   GGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGC

   TGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTAAAA

   GGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT

   TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC

   TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGT

   GGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG

   CAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT

   CAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC

   TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC

   GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG

   GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC

   CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA

   ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCC

   TGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG

   GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG

   CTGGCCTTTTGCTCACATGTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTT

   ACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC

   ACAGGAAACAGCTATGACCATGATTACGCCAAGCTACGTAATACGACTCACTATAGGG

   CAGATCTTCGAATGCATCGCGCGCACCGTACGTCTCGAGGAATTCCTGCAGGATATC

   TGGATCCACGAAGCTTCCCATGGTGACGTCACCGGTTCTAGATACCTAGGTGAGCTC

   TGGTACCCTCTAGTCAAGGCCTATAGTGAGTCGTATTACGGACTGGCCGTCGTTTTAC

   AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCC

   CCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACA

   GTTGCGCAGCCTGAATGGCGAATGGCGCTTCGCTTGGTAATAAAGCCCGCTTCGGCG

   GGCTTTTTTTT

10. Title this sequence as “Plasmid DNA” in the “Name of Sequence” box. Select “Circular” by the heading “The sequence is:”. Leave the other settings on their default status.
11. Click the “Submit” button (located to the right of the pasted DNA sequence).
12. On the new page that opens, select “Custom Digest” under the “Main Options” heading on the left side of the page.
13. The new page that opens will contain a list of restriction enzymes. Select the enzymes BamHI and EcoRI then click on the “Digest” button at the bottom of the page.
14. On the new page that opens you will see a circular representation of your DNA. Select “Fragments” under the “List” heading on the right side of the page.
15. Record the length of the longest fragment in Table 1. This fragment contains the majority of the plasmid DNA sequence. The short fragment is the excised plasmid DNA that lies in between the two restriction enzyme sites.