1. Restriction Analysis of Plasmid DNA

2. Session 1: Restriction Digest Reactions

3. Your sample is EITHER plasmid DNA pAMP or pKAN. Which do you have?
How could you tell them apart?
What’s different between them?

4. Each restriction enzyme cuts DNA wherever its recognition site appears.
Each restriction enzyme recognizes a particular sequence of nucleotides, called its restriction site. Many recognition sites are palindromes.
BamHI
…NNNGGATCCNNN… …NNNG GATCCNNN…
…NNNCCTAGGNNN… …NNNCCTAG GNNN…
HindIII
…NNNAAGCTTNNN… …NNNA AGCTTNNN…
…NNNTTCGAANNN… …NNNTTCGA ANNN…

5. A restriction map identifies where restriction sites appear along a DNA.
BamHI cuts here
HindIII cuts here
What will be different between the DNA fragments produced by cutting pAMP vs. pKAN with BamHI & HindIII?

6. DNAs can be distinguished from each other by restriction mapping.
3755 bp
2332 bp
784 bp
1875 bp
1904 – 1120 = 784
4539 – 784 = 3755

7. Glove Up! Put on a pair of lab gloves S, M, L, XL available
Most hands will fit in M or L gloves.
Try those sizes first unless you have particularly small or large hands.
Made of nitrile (no latex = no allergies)

8. Micropipetting Review
1st Step, 1st Stop.
2nd Step, 2nd Stop.
Micropipetting Review
FIRST STEP: Measuring
SECOND STEP: Dispensing
Choose a micropipette whose range spans the desired volume. Set the micropipette to the desired volume by turning the plunger knob. Don’t force past the pipette’s limits, as this breaks the pipette! Place a tip on the micropipette, matching tip and plunger colors. Depress plunger to the FIRST STOP and HOLD. Place the tip into the liquid. Slowly release the plunger, keeping tip in liquid.
Place tip against side of tube, near bottom. Depress plunger to the SECOND STOP and HOLD. Remove tip from tube while holding down plunger. Release plunger. Throw away used tip using the ejector button. Use a new tip each time you pipet.

9. Label a Restriction Digest Tube
From the jar with the white screw cap, remove one 1.5ml microtube. With a lab marker, label the lid of the microtube with your period number and the first initials of each team member. P1
TDH

10. Add Plasmid DNA
Your team was given a sample of either pAMP or pKAN plasmid DNA in a tube labeled “DNA” and a number between 1 and 12.
From this tube, use your micropipette to measure 5μl (microliters) of plasmid DNA and transfer it to your Restriction Digest tube.
At 0.1μg/μl,
this 5μl contains
0.5μg or 500ng of DNA.
DNA 1…12
5μl P1
TDH

11. Add Water
From the tube labeled H2O, measure 9μl of water and transfer it to your Restriction Digest tube.
H2O
9μl P1
TDH

12. Add Restriction Reaction Buffer
Enzymes require a chemical environment of the right pH and concentration of ions. The 5X restriction buffer is a concentrated mix that provides the environment needed for the restriction enzymes to work properly.
From the tube labeled 5X RE Buffer, measure 4μl of 5x Restriction Digest Buffer and transfer it to your Restriction Digest tube.
5X RE Buffer
4μl P1
TDH

13. Add Restriction Enzymes
You will cut your plasmid DNA with two restriction enzymes: BamHI and HindIII.
From the tube labeled BamHI + HindIII measure 2μl of the BamHI and HindIII mix and transfer it to your Restriction Digest tube.
BamHI + HindIII
2μl P1
TDH

14. Incubate the Restriction Digest Reaction
Close the cap on your Restriction Digest tube and place it in the heating block set at 37°C. The restriction enzymes work best at 37°C. The reactions will incubate for one hour, then be stored in a freezer until you examine them using gel electrophoresis.

15. Prepare the Restriction Digest Reactions
Agarose Gel Electrophoresis
4/12/2018
Prepare the Restriction Digest Reactions
Reaction Component
Volume to Add
Your Plasmid DNA Sample (
5µl
H2O
9µl
5X RE Buffer
4µl
BamHI + HindIII Restriction Enzyme mix
2µl
Total Volume
20µl
dNTP mix from Pitt Stockroom: 10mM for $58 -> dilute 1:5 to 2mM (add 800ul H2O) -> makes 80 x 12.5ul (2.5-reaction) aliquots

16. Session 2: Gel Electrophoresis

17. Prepare Your Samples for Loading
Agarose Gel Electrophoresis
4/12/2018
Prepare Your Samples for Loading
Add 4µl of the 6X Loading Dye to your restriction digest sample.
If your liquids are sticking separately to the side of the tube, flick the tube with your finger and tap the bottom gently on your lab bench, or spin briefly in microcentrifuge to collect entire sample at bottom of tube.

18. Load Your Sample On The FlashGel
When called, bring to the FlashGel:
Your DNA sample
Micropipette with tip
Load 6μl of your sample into a well.

19. Agarose Gel Electrophoresis
4/12/2018
Write your team initials or team number below the well into which you loaded your sample.
Lane 1 2 3 4 5 6 7 8 9 10 11 12 13
1kb ladder
Period ____
Teacher: Print out copies of this slide to place near each gel each period. Have students write their initials or team number in the lane into which their sample was loaded.

20. Run the Gel
A power supply provides current to the electrodes and through the buffer and gel. The progress of migration through the gel is monitored with tracking dyes that are visible without the transilluminator. 1.2% Flash Gel 200 V 8 minutes

21. Analysis of Gel Results

22. Restriction Mapping Can Be Used To Identify Unknown DNAs
3755 bp
2332 bp
784 bp
1875 bp

23. Agarose Gel Electrophoresis
4/12/2018
1kb ladder 1 2 3 4 5 6 7 8 9 10 11 12 13
Restriction Fragment Sizes
pAMP:
3755,
784
pKAN:
2332
1875
Promega BenchTop 1kb Ladder
Period #1
Teacher: After capturing a digital image with the Flash Gel software, return to this slide. Right-click on the gel image, choose “Change Picture…”, and browse to the gel image you just captured. That will replace this image with your new one.
1.2% 200V 8min

24. What Questions Do YOU Have?

25. Materials

26. Agarose Gel Electrophoresis
4/12/2018
100bp DNA Markers
Bio-Rad EZ Load 100bp Molecular Ruler
# ; $99
Ready to load
Invitrogen 100 bp DNA Ladder
# ; $99
In TE
Invitrogen TrackIt 100 bp DNA Ladder
# ; $114
Promega 100bp DNA Ladder
#G2101; $111
-20C RT
10 bands
100 – 1000 bp
16 bands
100 – 1500bp bp
600bp band 2x-3x brighter
11 bands
bp bp
500bp band brighter
1 µg/µl
100ng/µl x 5ul/lane =
500ng/lane
130ng/ul x 5ul/lane =
650ng/lane
50ug
500ul = 50ug = 100 lanes
250ul = 32.5ug = 50 lanes
These are the 100bp DNA ladders that are stocked in the Department of Biological Sciences stockroom.

27. Agarose Gel Electrophoresis
4/12/2018
1kb DNA Markers
Bio-Rad EZ Load 1kp Molecular Ruler
# # ; $84
Ready to load
Invitrogen 1kb DNA Ladder
# ; $158
In TE
Invitrogen TrackIt 1 Kb DNA Ladder
# , $98
Ready-to-load
Invitrogen TrackIt 1 Kb Plus DNA Ladder
# , $125
Promega 1kp DNA Ladder
#G5711; $111
-20C RT
15 bands
1 – 15kb
22 bands
1 – 12kb + 100bp-500bp
20 bands
0.1 – 12kb
Doublet 1650/2000
1 µg/µl
100ng/µl x 5ul/lane =
500ng/lane
100ng/ul x 5ul/lane =
250 ul = 250ug
500ul = 50ug = 100 lanes
These are the 1kb DNA ladders that are stocked in the Department of Biological Sciences stockroom.

29. Teacher Pre-Lab Preparation and Classroom Set-Up

30. FastDigest HindIII: 3 μl
Preparing DNA Samples
“DNA A”
1kb Ladder
200ng / 5μl
2-3 stations
“DNA B”
Lambda/HindIII
500ng / 5μl
2 stations
“DNA C”
100ng / 5μl
“DNA D”
pAMP/BamHI/HindIII
“DNA E”
pKAN/BamHI/HindIII
FlashGel: “load 5-20ng/band in 5ul/well”
We load 100ng/5ul/lane
3 lanes/gel
1 gel/period
6 periods/day
Prepare 3.0 μg
Stock Conc.100 ng/μl
pAMP DNA: 30 μl
H2O: 18 μl
10X FastDigest Bfr: 6μl
FastDigest BamHI: 3 μl
FastDigest HindIII: 3 μl
Final Rxn. Vol. = 60 μl
37° 30 min;
37° 5 min
Add 90 μl 1X TE
Final Conc. = 20 ng/μl
Load 100 ng = 5 μl
(Flash Gel recommends 5 μl / well)

31. Lambda () / HindIII DNA Markers
Bacteriophage Lambda’s genome is 48,502 bp. When cut with the restriction enzyme HindIII, eight fragments are released. /HindIII is a commonly used set of DNA size standards for electrophoresis. Which DNA sample is lambda/HindIII?

32. Plotting a Standard Curve for the DNA Markers
Plot on Semi-Log Paper
X-axis = distance migrated
Y- axis = DNA length in base pairs (bp)

33. Workstation Set-Up

34. Supplies
Boxes of S, M, L, X nitrile lab gloves 0.5ml microcentrifuge tubes Bottle of water for gels Styrofoam cups ice

35. Equipment
Microcentrifuges Vortex mixers FlashGel running tray with blue-light illuminator Power supply FlashGel camera Laptop computer with FlashGel software
Traditional electrophoresis tank with