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22 Cards in this Set
- Front
- Back
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In the plasmid lab, what three genes might possibly have been inserted into your plasmid in this experiment? What restriction sites surround each.
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E Coli: PFK
B Stearotherophlous Ribonuclease T1 |
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What two visible dyes allows you to visualize the range of distance that DNA travels while the gel electrophoresis is running? Which moves the shortest distance on the gel.
Plasmid lab |
Bromophenol Blue Shorter
Xylene Cyanol FF Faster |
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What specific type of gel electrophoresis will you use to separate DNA strands of different lengths? What percentage gel is run?
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Agarose gel electrophoresis
1% gel |
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What is the name of the plasmid in this experiment? To what two antibiotics is this plasmid resistant?
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pALTER-1
Ampicillin and tetracycline |
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Name two restriction enzymes you will be using this experiment. What is the recognition for each site?
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HIND III --> AGCT
BAMHI --> GATC |
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DNA will migrate towards which end of the terminal? Why?
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DNA will migrate to the positive terminal because DNA has a negative charge proportional to its length.
Shorter DNA molecules move the longest distance |
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Name the component of the cell neutralization solution responsible for precipitating proteins during the purification of your plasmid.
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1.32M Potassium acetate
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What specifically will be used as a molecular weight marker in your electrophoresis
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Lambda DNA BSTE II digested
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What molecule will allow you to visualize DNA bands on the gel with the use of UV radiation? What danger is posed by this molecule?
Plasmid lab |
Ethidium Bromide
Carcinogen |
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What two reagents are used to lyse the bacterial cells during the plasmid purification?
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NaOH and 1% Sodium Dodecyl Sulfate (SDS)
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What function is provided by the "cell resuspension solution", other than simply providing a buffer to which to solubilize the cells?
Plasmid lab |
Contains DNAse A, removes RNA from the cytoplasm
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What reagents help to generate an interaction between the wizard resin and plasmid DNA? How is DNA eluted from this resin? Explain
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7M Guanidine HCl.
Water soluble so pure water makes it swell. |
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What physical feature might make DNA strands appear artifically large on gel electrophoresis? What physical feature might make DNA strands appear artificially small on gel electrophoresis?
Plasmid lab |
Nicked/cut plasmid = shorter distance
Supercoiling = further distance (artificially smaller so it moves farther) |
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Why is it necessary to wait until all groups are finished with the experiment before beginning to load samples within the wells of the gel?
Plasmid lab |
DNA will tend to diffuse out of the gel
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Why is genomic DNA not co-purififed with the plasmid DNA by the wizard kit?
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TOo big to bind to the resin
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What reagents must be kept on ice during the plasmid experiment? Why?
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Restriction enzymes, so they don't denature
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Do shorter or longer pieces of DNA travel farther on agarose gel?
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Shorter pieces of DNA travel farther
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Do DNA samples need to be specially treated so that it moves through the gel based on molecular weight?
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No.
Double stranded DNA molecules have a negative charge based on their weight. |
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Towards which terminal do DNA samples migrate? Why?
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DNA moves towards the positive terminal because it has a negative charge proportional to its length
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If your sample exhibits supercoiling, what should you see?
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More then one band on the uncut plasmid.
Some plasmids supercoiled and traveled further |
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How do you calculate MWT from the graph in the plasmid lab?
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Graph plots log(Mwt) vs distance traveled.
You take the 10^log(mwt) |
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Which lane in the plasmid lab do you use to determine molecular weight of the phagemid?
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Lane 2 (HIND III) because dna material was cut out by restriction enzyme to produce pALTER-1 fragment clearly.
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