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 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

Gel Electrophoresis:• Pretty straightforward

+

-

Priming you on Primers

Determining Insert Size – pop quiz!

Minipreps

•Purpose•To purify the DNA for sequencing

Schematic OverviewSupernatant = just LB amp

Degrades RNA

Lysis buffer, NaOH;

denatures all double strands

Plasmids to

renature; net

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

1200 bp

400 bp

2900 bp

What happened?

Do they agree?

QUESTIONS?