Transcript Let*s play the review game!

LET’S PLAY THE
REVIEW GAME!
(a.k.a how much did you forget this summer?)
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??)
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
Million Dollar Question:
Which colony
should I pick??
“A” is for A+
• We pick white colonies for a reason
• Two kinds of plasmids present:
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
Everybody <3 PCR!
• Purpose?
• To amplify the insert DNA even further to
get a good band in a gel, like so:
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
Lightning Review!! PCR details
1. Initial Denaturation
1. 94˚C for 5 minutes
Lyse cells, separate DNA strands
2. Amplification
1. 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 Elongation
1. 72˚C for 5 minutes
Ensures all DNA strands are full length
-
Gel Electrophoresis:
• Pretty straightforward
+
Priming you on Primers
Determining Insert Size – pop quiz!
Minipreps
• Purpose
• To purify the DNA for sequencing
Schematic Overview
Supernat
ant = just
LB amp
Degrades
RNA
Plasmids
to
renature;
net
Lysis
buffer,
NaOH;
denatures
all double
strands
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?
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.
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
Example
2900 bp
1200 bp
400 bp
What happened?
Do they agree?
QUESTIONS?