Hold micropipet and epitubes at eye level - PowerPoint PPT Presentation

Title: Hold micropipet and epitubes at eye level


1
Hold micropipet and epitubes at eye level
2
Micropipet Use

• Add disposable pipet tip
• 2 Press plunger to first stop
• 3 Insert pipet tip into solution to be
  
• transferred
• 4 Slowly release plunger to retrieve liquid
• 5 Move pipet tip into desired tube
• 6 Press plunger past first stop to second stop
• to transfer liquid, keep the plunger down
• as you remove it from the tube.
• 7 Eject tip

3
Micropipetting technique A
4
technique B
Close the tips!
5
(No Transcript)
6
Micropipet tip should be ABOVE the well NOT IN
IT!!!!
7
Micropipet tip punched right through the gel See
dye under the wells
8
NICE!
9
When you to into the lab today, you will first
practice using the micropipette
Each person in your group will transfer 15 uL of
colored liquid into an empty microtube. Once this
is completed, you should have 60 uL of brownish
colored liquid in your practice tube.
15 uL
15 uL
15 uL
15 uL
10
After practicing how to use the micropipette, a
biotechnology student will demonstrate and load
the victims DNA into the gel.You will then
load your DNA into the gels.Each student in the
group can then load 1 well of the gel with the
suspects DNA.There are four suspects
total.You may NOT load the gel without the help
of a biotechnology student watching you!
11
To understand electrophoresis, you must
understand the structure of DNA
12
Phosphate groups of nucleotides have a - charge
5
5
1
3
OH
2
Phosphodiester bond
3
13
Charge on a DNA Double Helix
Large groove
Small groove
1 Twist 10.5 bp
14
Gel Electrophoresis

• Gel Electrophoresis technique uses the
  difference in electrical charge to separate
  polymers (DNA, RNA, protein) on the basis of size
• Lets see a model of how gel electrophoresis works

15
DNA Electrophoresis analysis after DNA has been
cut by enzymes at various points, A and B
Restriction enzymes
A B
10 kb
C A B AB L
Juang RH (2004) BCbasics
16
Purpose of Gel Electrophoresis?

• Get a DNA Fingerprint of individuals
• Trace a sequence of genetic markers in families
• Diagnose disease
• Compare genes of different people, different
  species
• Find mutations
• Paternity tests

17
Restriction Enzymes

• rDNA (recombinant DNA)the produced piece of DNA
  from inserted another piece of DNA
• recognize specific sites to cut the DNA
• Blunt endsstraight across
• Sticky endsone side of DNA is longer than the
  other, these overhangs allow for complementary
  matches between two DNA pieces cut by the same
  enzyme, the sticky ends match and pasting ma
  occur to produce an rDNA molecule
• More than 1200 restriction enzymes discovered
  isolated from bacteria
• Read 5? 3
• Palindromic (example radar or GAATTCCTTAAG

18
Reviewing Homework
19
Based on this DNA Fingerprint created, who should
the police arrest as the prime suspect? Suspect 1
or 2? Why?