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16 Cards in this Set
- Front
- Back
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What is SSC and why do we use it?
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Sodium chloride and sodium citrate
used to favor hydrogen bonding between probe and target as opposed to interactions with water molecules |
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Why do we use PCR after microsatellite isolation?
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to make the genomic DNA double-stranded (and many copies) for cloning into vector
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How do we elute the DNA away from the probe in microsatellite isolation?
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Heat and low salt conditions disrupt the hydrogen bonds while biotin stays attached to streptavidin on the beads
65˚C for 10 minutes in water (no SSC) |
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How do we utilize different concentrations of SSC?
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20X mixed with probe to make the probe anneal to DNA better
0.5X buffer to wash the beads |
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What are SA-PMPs?
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streptavidin-paramagnetic particles
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Compare and contrast the features of pBluescript and pCR2.1
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Both have: MCS, ori, selectable marker (pCR2.1 has Kan in addition to Amp), and LacZ (but we only use it in pCR2.1 as pBluescript was never used for transformation)
pCR2.1 has Topoisomerase |
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why do we spread IPTG and X-gal onto the LB-amp plates separately?
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if mixed, they can precipitate
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A colony lift...
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...is a way to transfer DNA from a bacterial colony or phage plaque to a nylon membrane and then immobilize it for further manipulation.
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A colony lift...
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...is a way to transfer DNA from a bacterial colony or phage plaque to a nylon membrane and then immobilize it for further manipulation.
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what are the steps to a colony lift?
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- touch nylon membrane to plate
- denature - neutralize - UV crosslink |
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What is in the denaturing buffer and why?
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1.5 M NaCl -- salt, helps negatively charged DNA bind to positively charged membrane
0.5 M NaOH -- cell lysis and makes DNA single-stranded |
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What is in the neutralization buffer and why?
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1.5 M NaCl -- continues to help DNA stick to membrane from denaturing buffer
1 M Tris base -- keeps DNA stable (reduces basicity after NaOH in denaturing buffer) pH 7.5 |
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Why do we do a hybridization screen if we have already purified for microsatellites?
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Microsatellite isolation is not 100% efficient, and we only want to miniprep the colonies that really contain microsatellites
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What are advantages to a library made from phage vectors?
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A phage vector can hold more DNA than a plasmid and phage plaques can be packed onto an agar plate at a much higher density (20,000)
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Where do we get streptavidin from?
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Streptomyces avidinii, which uses it to scavenge biotin (vitamin H) from its environment
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What are the steps of detection of hybridize probe (after UV crosslinking)?
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- incubate with biotinylated probe, EDTA, SDS overnight
- wash in 2x SSC/1% SDS - wash in 1x SSC/1% SDS - wash in 1x SSC (all pH 7) - block membrane with bovine serum albumin (BSA) Blocking buffer - Streptavidin-AP in Blocking buffer - wash in Blocking buffer - Wash buffer - Assay buffer pH 9.2 to make AP happy - CDP-Star AP substrate - X-ray film |
