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40 Cards in this Set
- Front
- Back
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DNA cloning |
Method of preparing well defined segments of DNA in multiple identical copies |
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How is DNA cloning done? |
By inserting the desired DNA into a bacterial plasmid and allowing the bacteria to replicate it |
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Recombinant DNA |
Resulting plasmid that contains the desired DNA |
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The plasmid acts as a... |
Cloning vector |
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Restriction enzymes |
Normally, they cut up foreign DNA to protect the bacteria |
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Each restriction enzyme recognizes a different... |
Restriction site that they cut |
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How the the DNA of a bacterial cell protected from its own restriction enzymes? |
Methyl groups are added to the sites that match up to the restriction enzyme's restriction site |
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How to make a recombinant DNA plasmid |
Use restriction enzymes to cut the plasmid where the cloning DNA should be inserted, then introduce cloning DNA to the plasmid (should have been cut by the same enzyme), and then use DNA ligase to seal |
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Restriction fragments |
Resulting fragments cut by a restriction enzyme - cuts the same fragments no matter what |
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Why can't PCR substitute for cloning? |
PCR has occasional errors - limits # of good copies of DNA wanted |
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In lab, what plasmid did we use? |
pJet plasmid |
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Features of pJet1.2 cloning vector |
Ampicilin resistance w beta-lactamase gene, eco47IR gene disrupted when desired DNA is ligated into the vector |
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What does eco41IR encode for? |
A restriction enzyme toxic to E.coli |
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What if the bacteria takes up a plasmid, but not a plasmid that ligated the desired DNA? |
ECO47IR will not be disrupted and the bacteria will be killed |
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Why does it not matter that the PCR product and the vector have blunt ends? |
We don't care about expression of the PCR gene, just the sequence |
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Why was it necessary to treat the PCR products with a proofreading DNA polymerase? |
To remove the 3' A overhang left from Taq's lack in proofreading ability |
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DNA ligation |
DNA ligase catalyzes formation of a phosphodiester bond between the 3' hydroxyl and 5' phosphate on the two DNA strands |
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Most commonly used DNA ligase |
T4 DNA ligase |
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Why is ATP added during ligation? |
5' end only has 1 phosphate group, so ATP needed |
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Results of vector self ligation |
Will die due to eco47IR effects |
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Ligation of a vector with primer dimers or other short PCR products |
Will survive |
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Ligation of a vector with multiple insertions in tandem |
Will survive, and are ok for our purposes (would be bad if they overlapped though) |
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Self-ligation of insert |
No ampicillin resistance - will die |
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How we transformed our bacteria |
Calcium chloride and heat shock |
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What does the calcium chloride do? |
Allows DNA to more readily associate with the bacterial cell surfaces |
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What is the heat shock presumed to do? |
Rapidly melts our breaks down cell membranes, allowing DNA in |
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"Satellite" colonies |
Small colonies that form around larger ones - only survive because the transformed cells already destroyed antibiotic in the vicinity |
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What kind of bacteria can exclusively be used in transformations |
Mutant strains with little to no restriction activity |
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What is the analytical digest used for in plasmid purification? |
Analytical purposes |
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What will happen in the digest? |
Restriction endonucleases (remove bases interior of DNA molecule) cut at the BglII sites and release the inserted DNA |
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Exonucleases |
Remove base pairs from the ends of DNA molecules |
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Plasmid purification to... |
Isolate plasmid DNA from gDNA |
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Step 1 in plasmid purification |
1. Alkaline lysis - cells treated with an alkaline solution to disrupt the cell membranes and denature proteins, cell bursts, DNA denatured |
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Components of alkaline solution in part 1 |
sodium hydroxide, sodium dodecyl sulfate (SDS), and ethylenediamine tetraacetate (EDTA) |
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Function of SDS |
Disrupts cell membranes and denatures proteins |
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Function of EDTA |
Chelating agent, removes Mg ions and destabilizes bacterial wall |
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Why do the plasmid DNA molecules remain intact? |
Small and supercoiled |
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Step 2 in purification of plasmid |
2. Neutralization - acid added - as pH drops, gDNA renatures and aggregates, high salt solution precipitates proteins - cellular debris and all this other stuff is centrifuged out |
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Final step in plasmid purification |
3. Remove salts and other contaminants - silica column chromatography used |
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How does silica column chromatography work? |
Phosphates on DNA adhere to the silica |
