Molecular Biology
Page 2: Goals of the Lab
Know the parts of electrophoresis apparatus
Understand how to use gel electrophoresis results to estimate DNA fragment sizes
Know how to reconstruct a plasmid based on a double digest gel image
Outputs:
Complete a restriction digest
Load a gel using a micropipette
Gel Electrophoresis Worksheet and Standard Curve Graph of pUC 19 Fragments
Reconstructed plasmid map
Page 3: Recombinant DNA technology
Use of restriction enzymes to insert genes from one organism into another
Restriction enzymes cut DNA at specific sequences called restriction sites
Restriction site sequences are palindromic and symmetrical
Page 4: RE nomenclature
Restriction enzymes are named after the bacteria they were isolated from
Example: EcoRI is named after E. coli, where it was discovered
Page 5: Recombinant DNA
Restriction enzymes and DNA ligase can be used to make recombinant DNA
Recombinant DNA is a combination of DNA fragments from different sources
Restriction enzymes recognize cleavage sites and create "sticky ends"
Complementary ends of introduced DNA fragment associate with sticky ends
DNA ligase catalyzes the formation of hydrogen bonds at sticky ends
Page 6: Recombinant technology in DNA analysis
Examples: DNA sequencing, expressing a gene in another organism, etc.
Constructing a map of DNA involves cutting DNA into smaller pieces, sorting them by size, and reconstructing the full DNA molecule
Page 7: Mapping plasmid DNA
Plasmids are small extrachromosomal circular DNA molecules found in yeast and bacteria
Use of two restriction enzymes (Ava II and Pvu II) and gel electrophoresis to map the pUC 19 plasmid
Page 8: Gel electrophoresis
Agarose gel is used to visualize the results of a restriction digest
DNA fragments are forced to move through the gel by electric current
Larger fragments get "stuck" while smaller ones move further
Electrophoresis buffer transmits the electric current to the gel
Page 9: Gel electrophoresis
DNA is negatively charged due to its phosphate groups
DNA migrates towards the positive electrode when electric current is applied
High current leads to faster movement, while low current leads to slower movement
Page 10: Gel electrophoresis
Agarose matrix is homogeneous
Ladder, a molecular weight standard, is loaded in each gel to compare fragments in the sample
Page 11: Digesting pUC19
Double digest of pUC 19 using two restriction enzymes (Ava II and Pvu II)
Incubate pUC 19 with REs, buffer, and water at 37C for 30mins-1hr
Tube A: 24ul H2O, 3ul Ava II buffer, 2ul pUC 19 DNA, 2ul Ava II RE
Tube P: 24ul H2O, 3ul Pvu II buffer, 2ul pUC 19 DNA, 2ul Pvu II RE
Page 12: Gel electrophoresis: staining
Loading dye is needed to visualize DNA and help the sample sink to the bottom of the well
Different dyes can be used, such as SYBR green, methylene blue, bromophenol blue, or ethidium bromide
Page 13: Gel electrophoresis procedure
Mix 5ul of each tube with 3ul of loading dye and load into individual wells on a gel
Also load uncut pUC 19 plasmid and two molecular weight standards
Run the gel for 45 minutes to 1 hour
Take a picture of the gel and analyze the distance migrated by each DNA band
Page 14: Standard curve of DNA fragment size
Use semi-log graph paper to plot molecular weight standards and distance migrated
Draw a best fit line through the data points
Use the standard curve to estimate the size of each restriction fragment
Page 17: Reconstructing the plasmid
Determine the number of DNA fragments made during each digest
Compare the number and position of the restriction sites on the circular plasmid
Page 18: Constructing a DNA map of the plasmid
The size of each DNA segment is known
Use the double digest fragments to create the larger fragments from each single digest
Page 19: Constructing a DNA map of the plasmid
Find which DNA segments of the double digest fit together to make the bigger restriction segments from each single digest