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16 Cards in this Set
- Front
- Back
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Why do we use microbes to clone genes? |
- We use micro oraganism because we can grow 1 Liter of micro-organism to give the same amount of gene for 1 human body genes. human = 2genes/cell E.coli = 500 genes/cell - Plus no one cares if u kill bacteria |
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What is a vector |
a type of DNA that is able to replicate itself inside a cell e.g. plasmid is a type of Vector |
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1. Recombinant Vectors (Plasmids): Plasmids have two essential ingredients to enable self-replication & maintenance.What are they? and What are their functions? |
1. ORIGIN OF REPLICATION: recruits DNA poly, is where replication originates. 2. SELECTION MARKER: so you can kill the plasmids that don't contain your gene of interest. 3. DNA cassette / insertion (optional) to express a biologic therapeutic.to express a multi-enzyme pathway.to express an engineered genetic circuit. |
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1. Recombinant Vectors (Plasmids): How does Origin of replication relate to the amount of plasmid replication that occur in each cell? |
example CoIE1 origin has a copy number of 40-60. This means if you break 1 cell of this bacteria, you will find 40-60 plasmid per cell. have negative feedback loop, so it stops at 40-60 copies per cell. Blocks the recruitment of the mechanism. |
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1. Recombinant Vectors (Plasmids): What does a selection marker contain? Why is it important to have this section in a plasmid vector? |
example: Tetracycline resistance(tetR), ampR, Kan. When antibiotics are added, only the cells with resistance selection marker will survive. Those cell are said to be "transformed." |
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1. Recombinant Vectors (Plasmids): What are Transformed cells? What are isogenic colony and satellite colony? |
Cells that are said to be transformed by plasmid are the cells that contain the gene that we want. They are cell that contain resistance gene inside their plasmid, therefore can survive when that certain antibiotic is added. ISOGENIC COLONY = the only cells that survive are only the ones we want. SATELLITE COLONY = when other cells survive too |
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2. DNA Purification / mini prepping: - what are the 3 steps in separating plasmid from cells? |
1. centrifuge to pellet your cells 2. add a strong base to open your cells and separate the DNA [all charge components are crushed out] 3. add a weak acid to neutralize The plasmid DNA re-assembles, but not the chromosomal DNA (too big!) [plasmid is small and comes back faster] |
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2. DNA Purification / mini prepping: what kind of solvent is used to purify DNA's? |
SILICA COLUMN, phosphate back bone sticks to the silica bc it's so hydrophillic |
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3. Enzymes to Chemically Modify DNA: What are the 5 important DNA's? |
1. Restriction Endonuclease - cuts DNA and makes sitcky ends 2. DNA Ligase - bind 2 frgments 3. T4 Polynucleotide kniase (PNK) - adds Pi to 5' end of DNA 4. DNA Phosphatase - remove Pi from DNA ends 5. Exonuclease - chew back sticky ends |
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3. Enzymes to Chemically Modify DNA: What is restriction endonuclease? how long is the recognition sequence and how do they read? |
binds to a specific DNA sequence, and cuts the DNA. can create “sticky ends”, later used for ligation. The recognition sequence is 4-8 base pairs long, and are always palindromic because they form dimers. |
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Restriction Digest: what are the ingredients needed? What is the temperature? For how long? |
0. Water (total volume at end is 50 uL)1. Restriction enzyme buffer (typically a 10X) 2. DNA (1 ug or more)3. Restriction enzyme (1 uL, or 10 Units) 37C, 6-9hours long |
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DNA Ligase: What does it do? What types of ends do they join? Which type is faster? how specific is ligation? |
- binds to two DNA fragments, and creates a covalent bond Two types of ligation reactions: - sticky end & blunt endDNA ends must have an extra phosphate (diphosphate) -sticky ends take faster to ligate than blunt ends. - Joining blunt with staggered ends ==> fill the staggered ends first and then join the blunt ends. - base pairing (annealing) must be perfect for ligation to happen. |
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T4 Polynucleotide kinase (PNK): |
adds a phosphate to the 5’ ends of DNA fragments. But when u order DNA you can choose if you want the 5' phosphate ends or not |
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T4 DNA Polymerase: which way does the polymerase fills? What is the special activity this polymerase has? |
- Polymerase activity (fill, 5’-->3’) - Nuclease activity (chew, 3’ exonuclease) |
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Polynucleotide Kinase (PNK): Why do we need this enzyme? Calf Intestinal PHOSPHATASE (CIP): What does it do and why is this enzyme useful? |
PNK: adds 5' phosphate group, which is needed for ligation onto the other sticky end. CIP: removes 5' Phosphate group, so fragments can't self ligate and can reduce the background of cloning |
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4. Gel Electrophoresis to Separate DNA by Size |
Gel Electrophoresis will separate your DNA fragments, so you can purify the one you want. |
