Genetics and Cell Biology 2081 Lecture Notes

Genetics and Cell Biology 2081 Lecture Notes





















17_Light Reactions.Oct12






23_Cytoskeleton_1. Microtubules.Nov12

24_Cytoskeleton_2. Microfilaments&Intermediate Filaments.Oct14
















41_Deletion Mapping.Hmwrk.Feb09




















58_Mutagenesis. Mar12


Genetics and Cell Biology 2081 Lab Handouts

Genetics and Cell Biology 2081 Lab Handouts














































Krebs Cycle with Molecular Models

Krebs Cycle with Molecular Models

The Krebs cycle (alias, the citric acid cycle, alias the tricarboxylic acid cycle), when reduced to its most fundamental purpose, generates reducing power in the form of NADH and FADH2. It does this by “dissecting off” hydrogens from two carbon fragments remaining after glucose goes through glycolysis and subsequent decarboxylation of pyryvate yielding acetyl coenzyme A. The acetyl group is fed into the cycle by attachment to oxaloacetate, yielding citrate.

Here is an overview of the molecules involved in the Krebs cycle


What follows are molecular models of the sequential molecules involved in the Krebs Cycle:

Oxaloacetate Acetyl CoA transfers its acetyl group to the number two carbonyl carbon via the methyl end forming citrate
Citrate Note that it has a tertiary alcohol which is not oxidizable.
Isocitrate The hydroxyl has been shifted so that it is now a secondary alcohol, and can be oxidized.
Alpha ketoglutarate When Isocitrate is oxidized, leading to the reduction of NAD+, it also is decarboxylated
Succinyl CoA In a reaction similar to the formation of acetyl CoA, ketoglutarate is oxidized, decarboxylated and a CoA attached. (Note that the coenzyme A moiety is indicated by a turquoise group
Succinate The thioester bond in succinyl CoA is hydrolyzed forming fumarate, with generation of GTP linked to the process.
Fumarate Succinate is dehydrogenated, forming trans fumarate with the concomitant reduction of FAD to FADH. (Why isn’t this molecule in the cis configuration? Anyone?)
Malate Water is added to fumarate, leading to the formation of a secondary alcohol.
Oxaloacetate The alcohol is oxidized (similar to oxidation of isocitrate), reducing NAD+ to NADH, forming oxaloacetate.

Isolation of Buccal Cell DNA

Isolation of Buccal Cell DNA

This procedure is used to isolate individual DNA to be used in future PCR probes. Completely non-invasive and straight forward, it is a simple method to isolate small amounts of DNA from buccal cells.

15 mL sterile polypropylene centrifuge test tube
two sterile 1.5ml Eppendorf tube
5mL pipettor + sterile tips
1000 uL micropipettor + sterile tips
200 uL micropipettor + sterile tips
clinical centrifuge, balance
1.5 mL test tube holder (> 10 holes)
boiling water bath in 1000 mL beaker

10 mL 0.9% saline aliquoted into the 15 mL centrifuge tube
package of beverage straws
0.5 mL 10 % Chelex resin beads in sterile dH2O

Saline centrifuge tube
1.5 mL Eppendorf for separating Chelex
1.5 mL Eppendorf for storage of DNA
Label a 15 mL polypropylene test tube and the top of a 1.5ml Eppendorf tube with your name and/or seat number.


Pour the 10 mL of saline solution into your mouth and vigorously swish against your cheeks for 10 seconds.
With a beverage straw, deliver the saline wash solution back into the labeled 15 mL polypropylene test tube.

Place your test tube, with others, in a balanced array in the clinical centrifuge. Centrifuge at 2000 x g for 10 minutes. (Top speed, setting number 7 on the tabletop clinical centrifuge.)

pelleted buccal cells spun down from saline

The cells form a firm pellet below the saline supernatant.


SAVE THE PELLET, DISCARD THE SUPERNATANT by decanting into the sink with running water, taking care not to disturb pelleted cheek cells at the bottom of the tube.


Drain all of the saline supernatant.

ADD CHELEX BEADS: Chelex is an ion exchange resin which removes polyvalent metal ions which might break down DNA during boiling or inhibit PCR reactions (next experiment).
Enlarge the aperture of the end of a 1000 uL pipetor tip (blue) by clipping off 2 mm from so that particulate matter will not stop it up. Use this prepared tip to resuspend the 10% suspension of Chelex resin beads by pipetting the beads in and out of the micropipettor. Before resin settles, pipet 500 uL of Chelex into the 15 mL tube containing your buccal cell pellet. Save pipet tip.


RESUSPEND CHEEK CELLS WITH CHELEX Using the same prepared blue tip, resuspend the cells in the pellet by pipetting in and out several times. (If the tip stops up, snip off 2 mm of the tip.)


Examine the suspension carefully to ensure that no visible clumps remain.


Using the same prepared tip, transfer a 500 uL aliquot of the cells and resin suspension to a clean 1.5 mL Eppendorf tube labeled with your name.

chelex treated cells 100 C for ten minutes

BOIL FOR 10 MINUTES: The cells are lysed and proteins denatured by exposing to 100 C for ten minutes: place your sample, along with other samples from the group, into a 1.5 mL floating test tube holder and float in a boiling water bath for 10 min.


Time the ten minutes in the boiling water bath.


CHILL ON ICE: After the heat treatment, transfer all samples to crushed ice.


SPIN DOWN CHELEX: Place your chilled sample, along with others, in a balanced array in a microcentrifuge and spin for 30 to 60 seconds at top speed.


The chelex precipitates along with the denatured protein. The DNA is in the supernatant.


SAVE 200 uL CLEAR SUPERNATANT: Use a fresh pipet tip to transfer 200 uL of the clear supernatant to a clean 1.5 mL Eppendorf tube labeled with your:
Seat Number
cheek DNA
Take care not to pick up any of the cheek cell debris or resin from the bottom of the tube. Store your sample for a few minutes or hours on crushed ice or for days at -20 degrees Centigrade until you are ready to proceed to Set up and run PCR reaction


Glycolysis/Fermentation with Molecular Models

Glycolysis/Fermentation with Molecular Models

“Glycolysis” strikes fear into many undergrad biology students because it presents them with an abstract series of reactions and molecules which are difficult to visualize and therefore incorporate into a coherant biochemical framework. This exercise has each student taking responsibility for a single molecule in the series, learning the following about it:

1) its structure
2) its precursor
3) the enzyme which created it
4) the enzyme which will act on it
5) the product of that action
6) the significance of the bond structure, particularly those involving phosphate. (Note whether phosphoester (low energy) or phosphoanhydride (high energy).)

They are then to describe these features to their fellow students in sequence. This strategy for teaching glycolysis has received many positive reviews from students who have used it.

See the bottom of the page for a key to the elements and directions for the construction of the models.

Here is the sequential listing of the molecules of glycolysis


1) Glucose

Glucose is both an aldose and a hexose. It enters the cell by diffusion, and the action of hexokinase holds it there.

Hexokinase transfers a phosphate from ATP to the number six carbon on glucose. This not only initiates glycolysis, but traps glucose in the cell since the ionic phosphate group makes diffusion out of the cell impossible without assistance.


2) Glucose-6-phosphate (G-6-P)

Phosphoglucoisomerase moves the carbonyl from the number one to the number two carbon, changing the molecule from an aldose to a ketose. This will free up the number one carbon for the phosphoryllation of the next step.


3) Fructose-6-phosphate (F-6-P)

Phosphofructokinase-1 is a critical enzyme in several ways. It transfers a phosphate from ATP to the number one carbon, thus placing ionic “handles” on either end (a PO4 on both the number 1 and number 6 carbons), allowing for the “breaking” of the molecule in the next step. This enzyme is allosterically inhibited by elevated ATP concentration.


4) Fructose-1,6-bisphosphate (F1,6bisP)

Aldolase splits fructose-1,6-bisphosphate into two pieces, dihydroxyacetonephosphate (DHAP, a ketone) and glyceraldehyde-3-phosphate of the next step.


5) Dihydroxyacetone phosphate (DHAP)

The image at the upper left shows fructose 1,6-phosphate at the bottom, splitting into dihydroxyacetone phosphate above on the left, and glyceraldehye-3-phosphate above on the right.

Triose phosphate isomerase moves the carbonyl from the number two carbon to the number one carbon, isomerizing DHAP to glyceraldehyde-3-phosphate. Thus, a single glucose generates two glyceraldehude-3-phosphates and all the following reactions are doubled.

The lower image shows dihydroxyacetone phosphate above on the left, and glyceraldehye-3-phosphate close up.


6) Glyceraldehyde-3-phosphate (3-GAP)

Glyceraldehyde-3-phosphate dehydrogenase performs a complex reaction in which glyceraldehyde-3-phosphate is oxidized by the removal of the hydrogen from the aldehyde. This hydrogen is used to reduce NAD+. A phosphate is added to the number one carbon in place of the hydrogen. This produces a very high energy phosphoacid anhydride.


7) 1,3-bisphosphoglycerate (1,3bisPGA)

Phosphoglycerokinase transfers the high energy phosphate from the phosphoanhydride bond on the number one carbon to an ADP (substrate level phosphoryllation).

Note that this is the first ATP to be generated, and two are created for every glucose molecule which entered the pathway.


8) 3-phosphoglycerate (3-PGA)

Phosphoglyceromutase moves the phosphate from the number three carbon to the number two carbon to prepare it for dehydration in the step after next.


9) 2-phosphoglycerate (2-PGA)

Enolase dehydrates 2-phosphoglycerate to form phosphoenolpyruvate.


10) Phosphoenolpyruvate (PEP)

Pyruvate kinase transfers the high energy phosphate from PEP to ADP, yielding pyruvate and ATP.

Phosphoenolpyruvate is the most energetic molecule in all of the molecules in glucose catabolism, because of the strain in the enol and the phosphate being adjacent to an ethylene bond.

It can engage in substrate level phosphoryllation, donating its phosphate to an ADP yielding ATP.


11) Pyruvate

Pyruvate is the end product of glycolysis, and, in the presense of oxygen, will be dehydrogenated by pyruvate dehydrogenase to yield acetyl coenzyme A, the “crossroads” molecule of carbon metabolism.


12) Lactic acid

Lactate dehydrogenase regenerates NAD+ (required for oxidation of glyceraldehyde-3-phosphate by (named for the opposite reaction) reducing pyruvate. This happens in muscle during intense exercise (ergo, muscle burn) and in milk during fermentation (butermilk and yogurt).


13) Acetaldehyde

Pyruvate decarboxylase decarboxylates pyruvate in yeast to produce acetaldehyde. Thiamine is required for decarboxylation, and yeast synthesizes it in large quantities, making nutritional yeast an excellent source for this vitamin.


14) Carbon dioxide & Ethanol

Alcohol dehydrogenase oxidizes acetaldehyde and concomitantly oxidizes NADH to yield ethyl alcohol and NAD+, required for oxidation of glyceraldehude-3-phosphate thus allowing glycolysis to continue in the absense of oxygen or other hydrogen acceptor.

Once students have constructed the assigned molecule, and learned the enzymes and related molecules, the models are laid out in sequence on a big table, and students in succession gave the following information:

1) The name of the molecule they had constructed
2) Its characteristic features
3) How it differs from the previous molecule
5) The enzyme which produced it
6) How it will be changed into the next molecule and why
7) The name of the enzyme which performs this change and the meaning of the name of the enzyme.

Students were to discuss selected phosphorylated molecules (i.e. glucose 6 phosphate, 1,3 bisphosphoglycerate, and phosphoenolpyruvate), naming and discussing the bonds by which phosphate is attached, and the relative energy content in each type (phosphoester, phosphoanhydride, resons for PEP’s unusual energy). The products of hydrolysis of these bonds was demonstrated and discussed.

Following the discussion, students leave the room, the molecules are randomized, and students return and identify the molecules as a way of demonstrating what they have learned.


Here is the key for the identities of the elements in the models

For the construction, remember that in straight chain illustrations of the chemical structures:
The carbon back bone is vertical with vertical bonds from each carbon projecting away from the observer.

The horizontal bonds project towards the observer.

Review the lab protocol on the Krebs cycle molecules.

Videos on glycolysis from Films for the Humanities and Sciences series which review the process are:

VIDT QH 633 .C45 1992 pt.1 Cell and Energy (Series Title-Cellular Respiration)
VIDT QH 633 .C45 1992 pt.2 Glycolosis 1 (Series Title-Cellular Respiration)
VIDT QH 633 .C45 1992 pt.3 Glycolosis 2 (Series Title-Cellular Respiration

Genetics and Cell Biology 2081

Genetics and Cell Biology 2081

Here are lecture notes for last year’s Cell and last year’s Genetics (to be consolidated in the future.)
Coded grades for Genetics/Cell Fall Semester 2012, listed by your UC “M” number will be available here at the end of the Semester.

1) Table of Contents
2) Syllabus: Genetics/Cell Biology 2081, (Mount in front of your text)
3) How to Take a Fankhauser Genetics/Cell Biology Course
4) Wordstems for Genetics/Cell, chronologically by quizzes and tests
5a) Meanings of Wordstems for Genetics/Cell Biology, cumulative, with meanings, Part 1
5b) Meanings of Wordstems for Genetics/Cell Biology, cumulative, with meanings, Part 2
6) Study Groups: Towards Effective Peer Education
7) Study Group Report Form
8) Instructions for completion of Genetics Homework
Volleyball anyone?
9) Table of Contents of Lab Schedule and Activities
10) Lab Activities: Schedule for Genetics/Cell 2081
11) Laboratory Notebook Procedure
12) Format Suggestions for Table of Contents
13) Use of Contact Paper for Mounting Handouts and Specimens
14) Notebook Illustrations
15) Sample First Notebook Grade Sheet , from previous year
16) Sample Second Notebook Grade Sheet, from previous year
17a) How to View a Slide; Evaluating Microscope
17b) Binocular Microscope: its Features and Care; Microscope Storage Grade Sheet
18) Cell Structure in a Leaf Cross Section
19) Cells:The Functional Units of Organisms
20) Cells Found in Tooth Scrapings
21) Protein Assay by Microbiuret: Standardization
22) Sample Layout of an Experiment (Protein Conc. in Unknowns: Microbiuret)
23) Spectrophotometer Use
24) Graph Construction
25) Sample Math Problems for Cell Biology
26) Displacement Pipetters: Their Care & Use ; Practice Using Pipetter’s Features
27) Enzyme Assay: Lactase
28) Reagents, Materials and Calculations for Lactase Enzyme Assay
29) Lactase: Comparison of Content in Brands
30) Lactase pH Optimum
31) Glycolysis/Fermentation with Molecular Models
32) Protocol for Lineweaver-Burk Plot: Lactase Kinetics
33) Krebs Cycle with Molecular Models
34) Isolation of Chloroplasts by Differential Centrifugation
35) Reduction of DCIP by Purified Chloroplasts
36) Electrophoresis Gels: Practice Preparing, Loading, and Running Gels
37) Electrophoretic Separation of DNA Fragments
38) Endonuclease Digestion of DNA
39) Isolation of Buccal Cell DNA

Table for recording data Chloroplast Reduction of Indophenol in Light
(This table is still under Semester required construction…)

LINKS:Here is an excellent page to explore three dimensional protein structure of the enzyme ribonuclease When the page comes up, (it may take a while to load), you can “grab” the molecule with the left button of the mouse and rotate the structure in space. Alpha helices will be very apparent, beta pleated sheets can be found with some searching.

Lactase pH Optimum

Lactase pH Optimum

Enzymatic activity is strongly dependant on protein conformation. Since pH determines whether an amino acid’s side chain is charged or not, and ionic interactions affect tertiary protein structure, pH has a pronounced effect on a protein’s conformation and therefore on its enzymatic rate. Typically, the maximum rate of action of an enzyme is found only when it is folded in a precise fashion. The pH which produces this precise folding is termed its pH optimum. An enzyme’s pH optimum may be determined by performing multiple assays, each identical except for the pH at which it is run. Graphic display of the resulting data (reaction rate versus pH) demonstrates the enzyme’s pH optimum. Here we will determine the pH optimum of the enzyme lactase.

In the preparatory stage of this experiment, an array of buffers have been formulated which cover the pH range to be tested. Typically this can be done by preparing two stock buffers (one acidic, the other basic) which, when mixed together in varying proportions, yield varying pHs. The two stock buffers which we will use are: 1) boric acid/citric acid and 2) Na 3PO4. Varying their ratios produces pHs ranging from approximately 2 to 9. For the preparation of these buffers and their proportions for desired pH, see Chemical Technicians’ Ready Reference Handbook, p. 656-657.

As in many enzyme assays, adjustments in concentrations and volumes may be needed for optimum results. Keep careful track of how you set up your experiment. Refer to the protocols on Lactase Assay and Reagents for Lactase Assay.

Materials (per team of two students)
Enzyme dilution for ten tubes:
final concentration = 0.1 units/mL made by
suspending a tablet to make 100 units/mL (9000 units/90 mL)
dilute 0.1 mL 100units/mL into 100 mL dH 2O (1:1000 dilution)
20 mM o -nitrophenyl–D galactoside (3.0 mL ONPG)
series of buffers of noted pH made from boric acid/citric acid + Na3PO 4 in varying ratios
4% K2 CO3

test tubes: 10 clean 13×100 mm in rack
displacement pipetters, 0.2 & 1.0 mL
repeater pipetter, 10 mL chamber, set on 0.8 mL
37C hot block, 13 mm holes
cuvettes in rack



  1. Add enzyme dilution (0.8 mL) of  down the side of each tube, using repeat pipetter.
  2. Add specified buffer (1 mL each) to its appropriate tube (You may use the same pipet tip if you progress in sequence up through the buffers for all ten tubes, blowing out and tipping off any clinging droplets. Cross contamination effects should be minimal.  Vortex each tube, holding tube near the top to wash down the sides.
  3. Pre-warm these tubes in a 37 C hot block for two minutes.
  4. Start the reaction at 15 second intervals:  add 0.2 mL ONPG, vortex, start a stopwatch with 1st tube, replace tubes in 37 C hot block.
  5. Stop the reaction after exactly 15 minutes by adding 1.0 mL 4% K 2CO 3 down the side of the first tube, vortex and remove from hot block. At  each 15 second interval, repeat 4% K 2CO3 addition for each of the successive tubes, mix and transfer to the test tube rack.
  6. Read the absorbency at 450 nm, record in your notebook.
  7. Graph results (according to proper graphing technique) and discuss.

Graph of the results we got Autumn 2002.


Histology of a Leaf Cross Section

Histology of a Leaf Cross Section

This lab is designed to be used as a means of reviewing the use of the binocular microscope.

Slide: Sun Leaf Pear, B 669a

Scan the entire leaf section using the 4x objective , note major visible features, especially vascular tissue bundles and leaf tissue structure. Find a well-defined vascular bundle (not the central vein), then rotate the 10x objective into place. Finally, examine the vascular bundle with the 40x objective. Note the various classes of cells which you can distinguish. Illustrate the 400x view containing the following labeled features which should be familiar to you from first year biology:
epidermis: epidermal cells, cuticle, stoma, guard cells
mesophyll: palisade parenchyma, spongy parenchyma, intercellular space
vascular tissues: vascular bundle, bundle sheath, xylem (large, few) and phloem (small and numerous


Cross section of pear leaf at the central vein, 40x


Cross section of pear leaf central vein, 100x


Vascular bundle, showing xylem (larger red cells) and phloem (indistinct small greenish cells) wrapped in bundle sheath.


Smaller vascular bundle with pallisade parenchyma and spongy parenchyma clearly shown.
Here is a labeled version of the image of the histology of the leaf.


guard cells guarding stoma at center bottom.


guard cells guarding stoma at center bottom.
Here is a labeled version of guard cells and tissues.


guard cells guarding stoma at center bottom

Protocol for Lineweaver-Burk Plot: Lactase Kinetics

Protocol for Lineweaver-Burk Plot: Lactase Kinetics

A Lineweaver-Burk Plot is a graphical display in which the inverse of the rate of an enzyme is plotted against the inverse of the substrate concentration. A great deal of information about the enzyme can be gained using this graphic device. This treatment of the data allows extrapolation of the enzymatic rate at infinite substrate which is equal to the maximum velocity (Vmax.). The intercept at the 1/[S] axis is equal to -1/Km. It also allows distinction between competitive inhibitors (which have identical 1/v intercepts but non identical 1/[S] intercepts as the uninhibited enzyme) and non-competitive inhibitors (which have differend 1/v, but identical 1/[S] intercepts). The following experiment produces the data necessary for the plotting of a Lineweaver-Burk plot.

Equipment per team of two:
eight labeled clean 13 x 100 mm tubes in rack
37C hotblock, 13 mm holes, preheated
2 x 25 mL beakers (reaction mix and K2 CO3)
125 mL flask with about 30 mL dH2O
pipetters: repeat pipetter (Rxn Mix)
1000 uL (for H 2O, ONPG and K 2CO3 )
200 uL (for ONPG)
250 mL beaker for used tips
spectrophotometer with cuvettes & wipettes

Reaction mix:                          1 team      5 teams
dH 2O                                          1.9 mL      3.8 mL
0.1 M PO 4 buffer, pH 7.0     10.0 mL    50 mL
lactase,30 FCC units/mL       100 uL      500 uL
aliquot out 1.2 mL with repeating pipetter (10 mL syringe, refilled)
3.5 mL 20 mM ONPG (colorless) in 13×100 mm test tube
4% K 2CO3 : 12 mL in 25 mL beaker


Lineweaver-Burk Plot Experiment Table
Lineweaver-Burk Plot Experiment Table


Experiment Table:
Enter this table into your notebook, then number your tubes, set up in a rack, add the ingredients as specified in the steps below. Fill in A 450 and calculations as you work thru expt. You should also set up an additional set with a putative inhibitor (see bottom of page).


  1. Prepare reaction mix in 25 mL beaker, mix thoroughly. [Or use class prep]
  2. Add dH2 O to labeled tubes: the volume required to  q.s. to a final vol. = 2.0 mL.
  3. Deliver 1.20 mL of Rxn Mix to each tube.
  4. Prewarm to 37C for two minutes.
  5. At regular intervals (30 sec or 1 min), carefully add appropriate volume of ONPG to start reactions, mix, return to 37C. For small volumes of ONPG, take care to follow correct micropipette technique, especially do not dip too far into ONPG reagent, deliver to the side of the reaction tube just at but not touching the surface (avoid the possibility of picking up lactase and taking it back to the ONPG). Wipe pipet tip with wipette to clean between tubes. Vortex the tube to pick up all of the ONPG which may have been deposited on the side of the tube.
  6. At 15 minutes, add 1 mL of 4% K 2CO3 and mix at the same regular intervals to reaction tubes to halt the reaction. (Each tube should be incubated  exactly 15 min.)
  7. Read A450 for each tube against the blank tube (contains no ONPG).
  8. Graph on a linear scale to produce a  Substrate Saturation Curve. Here is an example of a linear/linear graph of activity versus substrate concentration.
  9. Fill in the table of the values for the inverses of substrate concentration and of enzyme velocities.
  10. Graph to produce Lineweaver-Burk plot. What is the V max? What is the K m? Here is an example of a double recipricol Lineweaver-Burke plot of the same data as above.

Inhibition experiment: Add final conc of 25mM glucose (0.5 mL of 1.0 M soln /12 mL Rxn Mx), or 5mM galactose (0.1 mL of 1.0 M soln /12 mL Rxn Mix) to test for possible inhibition of ONPG digestion. What kind of inhibition, if any, is demonstrated?  (19.8% = 1 M glucose = 1 M galactose).

Endonuclease Digestion of DNA

Endonuclease Digestion of DNA

Endonucleases are the highly specific enzymes which recognize unique palindromic sequences in DNA at which they hydrolyze the phosphoester linkage. These enzymes have been crucial in the burgeoning fields of genetic sequencing and engineering. Because endonucleases are commonly found on the surface of the skin, as well in bacteria, latex gloves are routinely worn and good sterile technique is required when performing DNA manipulations.

We will perform three digestions on purified lambda phage DNA:
1) with Hind III (A|AGCTT), isolated from Hemophilus influenzae,
2) with Eco RI (G|AATTC), isolated from Escherichia coli, and
3) with both. [BamHI (G|GATCC) may also be used]

The resulting fragments can be used to construct a restriction map of the lambda genome.

All equipment and supplies listed in the protocol on Electrophoresis of DNA Fragments
37C incubator
Microcentrifuge tubes

Lambda DNA 0.5 ug/µL [50 ug]
(We have found it convenient to purchase lyophyllized
lambda DNA and rehydrate it when needed.
Eco RI endonuclease, 10 units/µL [2000 units]
Hind III endonuclease, 10 units/µL [2000 units]
specific buffers for Hind III and Eco RI
Hind III lambda digest, 0.125 ug/µL
loading dye

1. Prepare the digestion tubes in an Eppendorf microcentrifuge tube:

Single digestion:

0.0 µL ddH2O (enough to q.s. to 30 µL, depending on DNA conc.)
3.0 µL 10x buffer for the endonuclease you are using
17.0 µL Lambda DNA (at 0.286 ug/µL, = 5 ug DNA)
10.0 µL Eco RI or Hind III (10 units/µL = 100 units total enzyme)
total volume: 30.0 µL
Set up the double digestion:

0.0 µL ddH2O (enough to q.s. to 30 µL, depending on DNA conc.)
1.5 µL 10x buffer for Eco RI
1.5 µL 10x buffer for Hind III (It is probable that these two buffers are interchangeable.)
17.0 µL lambda DNA (at 0.286 ug/µL, = 5 ug DNA)
5.0 µL endonuclease (10 units/µL = 50 units of enzyme A)
5.0 µL endonuclease  (10 units/µL = 50 units of enzyme A)
total volume: 30.0 µL

2. Incubate at least 30 minutes at 37C, an hour might be OK, perhaps needed for double digestion.

3. Halt the reaction by placing on ice, add 6 µL of 6x loading dye, mix with same pipet, reset volume to 25 µL for next step (leave the tip in the Eppendorf tube used for digestion).

4. Load the wells: Pipet 5 µL into one well, reset pipet to 25 µL, pipet that volume into second well. On the same gel, run a lane with 1 ug undigested lambda DNA, and one with 5 ug of a Hind III digest as a standard.

Run the gel, photograph or carefully diagram the resulting bands.

Here are results of a 2006 experiment to test for optimum quantities of enzyme and optimum quantities of DNA.

Below is the gel which we produced Winter Quarter 2005.  Problems?  Of course…  lanes 3 and 12 (BamHI) should be identical.  12 appears to have been contaminated with another endonuclease.  Lane 4 shows some double digestion, but may not have been digested long enough.  Lane 7 and 13 (EcoRI) should be identical.  Lane 7 did not have sufficient endonuclease activity.   But, endonuclese activity was demonstrated, and evidence of double digestion developed  on all lanes with double enzymes (lanes 4, 9, and 14).

electrophoresis of double digestion, 2005

Click here to see a similar experiement from 2009.

Below are less successful results from 2003. Some browsers resize the image, so be certain that you are referring to the correct lane. Clearly there were several problems. We used two different batches of endonucleases which may explain why digestion was observed in some cases but not in others. The second from the top may be smeared because the last of the enzyme was rinsed out of the tube in order to get the last bit for the digestion.

2003 endonucleases results

There are eight Hind III fragments: 23,130; 9416; 6557; 4361; 2322; 2027; 564;125 bp long.
The 564 band is in the bromphenol blue, and the 125 band appears beyond the dye band.

There are five Eco RI fragments: 21,226; 7421; 5804; 5643; 4878; 5330

The following fragments were reported on the web as the double digestion products:
Double: 21,226; 5148; 4973;4268; 3530; 2027; 1904; 1584; 1375; 947; 831; 564

prep of reagents for electrophoresis
eppendorf tube
DNA electrophoresis
Gel diagram
50x TAE buffer:

For 100 mL of 50x buffer:

24.2 g TRIS (base)
5.71 mL glacial acetic acid
10 mL 0.5 M EDTA (14.6 g/100 ml)