let’s play the review game! (a.k.a how much did you forget this summer?)
TRANSCRIPT
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LET’S PLAY THE REVIEW GAME!(a.k.a how much did you forget this summer?)
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LIGHTNING REVIEW!! ONs
•What’s an ON?•Why do we do ONs?
• (in other words, why is Vershon making us do manual labor when he’s already got the cDNA sequence??)
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Were you right?• Purpose of an overnight is to amplify cDNA (for use in
minipreps) – transforming plasmids with inserts into bacteria is a convenient way to get clones of specific insert
• Vershon can’t sequence the cDNA directly because…• He needs more copies• He needs to make sure the cDNA insert is worth sequencing
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Million Dollar Question:
Which colony should I pick??
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“A” is for A+• We pick white colonies for a reason • Two kinds of plasmids present:
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Then what?• Suspend colonies in 2mL of LB Amp (ampicillin to select
for transformed bacteria)• Incubate overnight at 37˚C to allow bacteria to proliferate
• Vigorous shaking to aerate culture and maintain max surface area
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Everybody <3 PCR!
• Purpose?• To amplify the insert DNA even further to get a good band in a gel, like so:
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2π is the magic number!• …Actually it’s 360 bp. If an insert is <360 bp long, it’s not
worth sequencing.
• What you need• Nucleoside Triphosphates• Forward and Reverse Primers• Taq Polymerase• The DNA
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Lightning Review!! PCR details
1. Initial Denaturation1. 94˚C for 5 minutes
Lyse cells, separate DNA strands
2. Amplification1. 94˚C for 30 seconds
2. 52˚C for 30 seconds (anneal primers)
3. 72˚C for 1 minute (nucleotides added)
4. Rinse, Lather and Repeat … 30 times
3. Additional Elongation1. 72˚C for 5 minutes
Ensures all DNA strands are full length
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Gel Electrophoresis:• Pretty straightforward
+
-
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Priming you on Primers
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Determining Insert Size – pop quiz!
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Minipreps
•Purpose•To purify the DNA for sequencing
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Schematic OverviewSupernatant = just LB amp
Degrades RNA
Lysis buffer, NaOH;
denatures all double strands
Plasmids to
renature; net
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Restriction Digests • Purpose: To verify the size of the insert• Restriction enzymes = molecular scissors that cut the
DNA at specific sites • We use restriction enzyme PvuII; two sites where PvuII
can cut:
• How many bands would you expect to see in the uncut lane?
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Digest this!
10.0 µl of DNA
The two most important rules in enzymes•Always keep enzymes on ice or in a cooler.
•Always use a fresh tip when pipetting from the enzyme stocks.
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Verifying the Size of the Insert• We know the size of the plasmid vector:
•2900 bp • We know the total bp upstream and downstream the
insert after cutting
•700 bp• Rule of thumb – subtract 700 from the smallest band on a
gel
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Example
1200 bp
400 bp
2900 bp
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What happened?
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Do they agree?
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QUESTIONS?