Stage 1: Prepare your plasmids to be cut by restriction enzymes

• Obtain the plasmids (pKAN and pAMP)P stands for plasmid

pKAN = plasmid with antibiotic kanamycin resistance

pAMP = plasmid with antibiotic ampicillin resistance

• Mix plasmids with restriction enzymes– BamH1 and Hind III– Restriction enzymes cut the plasmids at precise

locations

Restriction enzyme: Hind III

(cuts @ bp 234)

Restriction enzyme: Bam HI

(cuts @ bp 2095)

Plasmid w/ kanamycin resistance (pKAN)

pKAN = 4194 bp

1861 bp restriction fragment2333 bp

restriction fragment

Thousands of plasmids in our samples

K+(digested plasmid)

+ means the restriction enzymes were added

K–(uncut plasmid)

- means the restriction enzymes not added

How many plasmid fragments? How many plasmid fragments?

Stage 2: Check to see if the restriction enzymes worked

• DNA electrophoresis– Plasmid fragments are loaded into a gel– Connected to a power supply– Separates fragments based on their sizes– Smaller fragments travel further through the gel

Electrophoresis: sizes DNA fragments

10,000bp

8,000bp

6,000bp

5,000bp

4,000bp

3,000bp

1,500bp

1,000bp

Electrophoresis: sizes DNA fragments

Kno

wn

DN

A

mar

kers

K+

K-

A+

A-

2,000bp

500bp

Look at the lab handout and let’s predict the A+ fragments

2008 Data/Results

We will then micropipette the plasmids

Hold micropipette and tubes at

eye level

Micropipette UseMicropipette Use1. Twist dial to desired volumeTwist dial to desired volume2. Add disposable pipette tip. Add disposable pipette tip3. Press plunger to first stop . Press plunger to first stop 4. Insert pipette tip into solution to be . Insert pipette tip into solution to be transferredtransferred5. Slowly release plunger to retrieve liquid. Slowly release plunger to retrieve liquid6. Move pipette tip into desired tube. Move pipette tip into desired tube7. Press plunger past first stop to second stop. Press plunger past first stop to second stop to transfer liquid, watch liquid stick to wall to transfer liquid, watch liquid stick to wall of tube. Remove tip, then release plunger.of tube. Remove tip, then release plunger.8. Eject tip8. Eject tip

Micropipette UseMicropipette Use1 Twist dial to desired volumeTwist dial to desired volume2 Add disposable pipette tipAdd disposable pipette tip3 Press plunger to first stop Press plunger to first stop 4 Insert pipette tip into solution to be Insert pipette tip into solution to be transferredtransferred5 Slowly release plunger to retrieve liquidSlowly release plunger to retrieve liquid6 Move pipette tip into desired tubeMove pipette tip into desired tube7 Press plunger past first stop to second stopPress plunger past first stop to second stop to transfer liquid, keep the plunger downto transfer liquid, keep the plunger down as you remove it from the tube.as you remove it from the tube.8. Eject tip8. Eject tip

Loading Gels:

Insert pipette tip:

•Under buffer level

•Above gel well

Load the plasmids into an electrophoresis chamber

Micropipet tip should be ABOVE

the well NOT IN

IT!!!!

Micropipette tip punched

right through the gel

See dye under the

wells

NICE!

Connect the electrophoresis to a power supply…DNA has a negative electric charge.

So now what???

• Plasmids have been engineered for human uses. The human gene for insulin (red) can now be added to the plasmid. The bacteria will produce insulin for diabetics!

So now what???

The bacteria with the recombinant DNA replicates, thus passing the insulin gene onto its offspring. Each cell now will produce insulin for humans to harvest and use.

10,000bp

8,000bp

6,000bp

5,000bp

4,000bp

3,000bp

1,500bp

1,000bp

Mar

ker

DN

A

K+

K-

A+

A-

2,000bp

500bp

Mar

ker

DN

A

K+

K+

K+

A+

A+

A+

10,000bp

8,000bp

6,000bp

5,000bp

4,000bp

3,000bp

1,500bp

1,000bp

Mar

ker

DN

A

K+

K-

A+

A-

2,000bp

500bp

Mar

ker

DN

A

K+

K+

K+

A+

A+

A+