Download DNA Patterns

DNA Patterns
Introduction
In recent years scientists have used restriction analysis to help further our knowledge
about the structure of DNA, for mapping and sequencing DNA, and also for DNA typing for
identification purposes. Restriction analysis has three parts: DNA digesting, electrophoresis, and
staining plus analysis.
In the digest step, restriction enzymes recognize a specific base sequence within the DNA
and cut at that recognition site; this is the “digest” step. In this laboratory, we will use DNA of
the plasmid pAMP, a circular piece of DNA, with a total of 4539 base pairs, or we will use DNA
of the bacteriophage Lambda. The DNA will be already cut with three different restriction
enzymes: EcoR1, HindIII, and BamH1 (see restriction map on last page). Using a single enzyme
to cut DNA with multiple recognition sites for that enzyme will result in several fragments of
different lengths. See the maps of pAMP DNA and Lambda DNA with the expected fragment
sizes for each restriction enzyme.
5’…G^AATTC…3’
3’…CTTAA^G…5’
This DNA sequence above is the six-base sequence recognized by the restriction enzyme EcoRI,
derived from the bacterium Escherichia coli strain RY 13. The diagram indicates that the EcoRI
enzyme makes one cut between the G and A in each of the DNA strand so that after cutting, the
DNA is cut into two pieces:
5’…….G
AATTC.….…3’
3’….…CTTAA G……..5’
Note that the cut is not clean across one site in the DNA, but rather single-stranded tails are left
on each end, so-called “sticky ends” because they will rejoin readily by base-pairing rules.
Here are two other restriction enzyme recognition sites, to be used in our experiment; they also
generate sticky ends.
HindIII
5’…A^AGCTT…3’
3’…TTCGA^A…5’
BamH1 5’…G^GATCC…3’
3’…CCTAG^G…5’
In the second step of a restriction analysis, the mix of DNA fragments is subjected to
agarose gel electrophoresis to separate the DNA fragments by size. A gel made of agarose (a
type of purified agar) is made with wells to contain the sample, and the gel is placed in a
chamber with buffer solution. DNA fragments are loaded into the wells and when the current is
applied, DNA with its overall negative charge will migrate toward the positive pole (run to red).
As the DNA moves through the tangled pores of the agarose fibers, the smaller pieces move
faster, the larger pieces more slowly.
In the third step, the current is halted, the gel removed from chamber and stained to
visualize the DNA in the gel.
In your laboratory experiment, step 1, the digest, will be done prior to your lab, and you
will carry out steps 2 and 3: electrophoresis and visualization/analysis.
Materials and Supplies
electrophoresis chamber
power supply
micropipettor and tips
agarose for gel making
staining and destaining containers
TBE buffer
methylene blue stain (0.025%)
Per lab group:
on ice:
A = uncut pAMP DNA in 1.5 ml tube with loading dye
EB = pAMP DNA digested with EcoRI and BamHI in 1.5 ml tube with loading dye
EH = pAMP DNA digested with EcoRI and HindIII in 1.5 ml tube with loading dye
BH = pAMP DNA digested with BamHI and HindIII in 1.5 ml tube with loading dye
U = Unknown in 1.5 ml tube with loading dye
For DNA Patterns Lambda DNA (AP Bio Lab 6B version):
L/E = Lambda DNA digested with EcoR1 with loading dye
L/H=Lambda DNA digested with HindIII with loading dye
L=uncut Lambda DNA
Procedure:
Making Gels:
Make 0.8% Agarose Gel in casting tray for each lab group – can be cast a day or two before use
and moistened with TBE and kept in refrigerator wrapped in plastic wrap, or in ziplock bags.
To make 0.8% agarose gel – makes 200 ml.-sufficient for pouring 8 gels.
1. Add 1.6 g. to 200 ml of 1X TBE buffer in 600 ml beaker or Erlenmeyer flask.
2. Stir to suspend agarose.
3. Heat uncovered in microwave until dissolved – watch carefully. Can also use boiling water
bath to dissolve agarose.
4. Swirl solution and check that all agarose is dissolved.
5. Allow to cool to about 60oC before pouring – pouring too hot will damage casting trays.
The Electrophoresis Run.
1. Obtain your gel chamber and power supply. Check with your instructor to see if you will be
sharing a gel with another group.
2. Obtain agarose gel in tray and place in chamber so that the end of the gel with the wells is
closest to the negative end (black). (“DNA runs to red”)
3. Pour TBE buffer into chamber to fill two reservoirs and a little more so that you just barely
cover the surface of the agarose gel.
4. Obtain your five tubes, and prepare to load the samples into the wells. Skip the end wells if
you have enough wells.
5. Following the scheme drawn below, use a micropipet with a tip to load 10 l from each
reaction tube into a separate well of the gel. Use a fresh tip for each sample.
a. Steady the pipet over the well, using two hands.
b. Center the pipet tip over the well, dip the tip in only enough to pierce the buffer surface, and
gently depress pipet plunger to expel the sample. The loading dye in the sample has sucrose or
glycerol in it to make the sample heavy so that it will sink into the well. Be careful not to enter
into well because you don’t want to poke a hole in the bottom of the well.
6. Close the top of the electrophoresis box, and connect the electrical leads to the power supply –
red to red, black to black. Make sure that both electrodes are connected to the same channel of
the power supply.
7. Turn the power supply on – use 125 volt setting. Current flow can be confirmed if you observe
gas bubbles released from the electrode wires near bottom of chamber reservoirs.
8. Electrophorese for 40-60 minutes. Good separation will have occurred if the bromphenol blue
bands have moved 40-70 mm from the wells. If time allows, electrophorese until blue bands are
near end of gel, but be sure to stop electrophoresis before the blue band runs off the end of the
gel.
9. Turn off power supply, disconnect leads, and remove top of box.
10. Carefully remove casting tray from box, and slide gel from tray into shallow staining tray.
11. Flood gel with 0.025% methylene blue solution, and stain for 20-30 minutes.
12. When staining is complete, decant methylene blue solution back into storage container. Rinse
gel in running water, let it soak for several minutes in several changes of tap water. DNA bands
will become increasingly distinct as gel destains.
13. View gel over light box, then photograph for documentation.
*Results
For Lambda gel result, go to this link.
Here is a typical gel result using plasmid pAMP DNA:
pAMP E/B E/H H/B
The first lane contains uncut plasmid DNA and you see more than one band because the plasmid
can take many forms, and the form that runs fastest in the gel is the supercoiled circular form.
1. Look at the map of the pAMP plasmid on next page, and
 determine how many and what size fragments occur when pAMP is cut with EcoR1 and
BamH1: ______________.
 how many and what size fragments occur when pAMP is cut with EcoR1 and HindIII:
 how many and what size fragments occur when pAMP is cut with HindIII and BamH1:
2. In the space below, draw your gel results or attach your photograph of the gel.
3. Compare your fragment patterns to the patterns of the ideal gel. Account for differences.
4. What restriction enzymes were used to cut pAMP in the unknown sample – how do you
know?
MAP OF PLASMID pAMP
Map of Lambda DNA:
Questions
1. Define restriction enzyme:
2. What are restriction enzymes used for?
3. What is electrophoresis?
4. What is a restriction digest?
5. After electrophoresis, where are the smallest DNA fragments located in comparison to the
largest DNA fragments?
6. Look at the restriction map for Lambda DNA, and given that the DNA is 48,502 bps long,
calculate the size and number of DNA fragments created when the lambda DNA is cut with
EcoR1 and then again with BamH1. Fill in the chart below with fragment sizes. The calculations
are given for HindIII.
Lambda DNA cut with:
HindIII
23,130 bps
9416
6557
4361
2322
2027
564
125
EcoR1
BamH1
Go to this link to get instructions and practice on using gel measurements to estimate molecular
sizes of DNA.
Bonus Questions:
1. Using the restriction map of pAMP, predict what size fragments you would get if you digested
pAMP DNA with three restriction enzymes all at once, EcoRI, HindIII and BamHI?
2. Using the restriction map of Lambda, predict how many and what size fragments you would
get if you digested lambda DNA with both EcoR1 and BamH1 at same time.
*ANSWER Key available to Teachers – request by emailing [email protected]
Details of Prep for Teachers
Prepare:
0.8% Agarose Gel in casting tray for each lab group – can be cast a day or two before use and
moistened with TBE and kept in refrig. wrapped in plastic wrap, or in ziplock bags.
Recipe for 0.8% agarose gel – makes 200 ml.
1. Add 1.6 g. to 200 ml of 1X TBE buffer in 600 ml beaker or Erlenmeyer flask.
2. Stir to suspend agarose.
3. Heat uncovered in microwave until dissolved – watch carefully. Can also use boiling water
bath.
4. Swirl solution and check that all agarose is dissolved.
5. Allow to cool to about 60oC before pouring – pouring too hot will damage casting trays.
Each lab group should get on ice:
1.5 ml A tube containing 10 l of pAMP DNA loading dye
1.5 ml EB tube containing 10 l of pAMP DNA digested with EcoRI & BamHI with loading dye
1.5 ml HB tube containing 10 l of pAMP DNA digested with HindIII and EcoR1 and loading
dye
1.5 ml BH tube containing 10 l of pAMP DNA digested with BamHI & HindIII and loading
dye
1.5 ml U tube containing 10 l of Unknown (use E, or H, or B) and loading dye
Final mass of DNA in each tube should be about 1.5 g total; make up rest of 10 l volume with
1 l of loading dye and rest with distilled water.
0.2% Methylene Blue Stock Solution
Makes 100 ml; store at RT indefinitely.
Add 0.2 g of methylene blue to 100 ml of distilled water; stir till dissolved.
0.025% Methylene Blue Staining Solution
Makes 500 ml; store at RT indefinitely.
Add 62.5 ml of 0.2% methylene blue solution to 437.5 ml of distilled water. Stir to dissolve.
TBE Buffer, 10X. Makes 1 liter. Store at RT indefinitely.
1 g. NaOH
108 g. Tris base (mw 121.10)
55 g boric acid (mw 61.83)
7.4 g EDTA (disodium salt, mw 372.24)
Add all dry ingredients to 700 ml deionized or distilled water in 2 liter flask.
Stir to dissolve, use magnetic stir bar.
Add deionized/distilled water to bring to total of one liter.
1X TBE
Add 1 volume 10X TBE to 9 volumes of deionized/distilled water. Mix well.