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Laboratory Manual
Spring 2015
Dartmouth College
Department of Chemistry
Table of Contents
Introduction ....................................................................................................................... 1
Week One........................................................................................................................... 4
Week Two ........................................................................................................................ 18
Week Three...................................................................................................................... 24
Week Four ....................................................................................................................... 30
Week Five ........................................................................................................................ 35
Week Six .......................................................................................................................... 43
Week Seven ...................................................................................................................... 45
Week Eight ...................................................................................................................... 45
Lab Paper Guidelines ..................................................................................................... 46
Appendix 1: Halliwell et al. paper ................................................................................. 47
ii
Introduction
Biochemistry has greatly evolved as a research science over the past 20 years. Historically,
biochemists were scientists who obtained animal tissue (muscle, liver, brain, etc.), ground it
up in a biological buffer, and then proceeded to fractionate the soluble proteins using
chromatography in order to see which fractions contained specific activities. Eventually, the
fractionation resulted in a single protein, which could hopefully be assigned a specific
activity. This is how many of the metabolic enzymes were discovered and characterized.
While a fair amount of grinding still goes on in biochemistry labs, these days the sacrificial
organisms are somewhat smaller: usually bacteria, yeast, or stable eukaryotic cell lines. This
makes life for the biochemist much easier.
For the lab section of this course, we will start working at the DNA level, and continue all the
way up to the level of characterizing the encoded proteins specific activity. This is what
biochemists do these days. Unfortunately, science is a somewhat random process, subject to a
lot of variables that are relatively hard to control for. In short, research science is hard. While
one is expected to enjoy banging ones head against a wall for a few years during graduate
school (its character building), an undergraduate lab, with its obvious time and experience
constraints, demands something a little more predictable. This generally results in a
cookbook lab, which, while informative, does not necessarily teach you what it is like to do
research. So, we scoured the scientific literature for a system that was (practically) foolproof,
and involved an enzyme that 1) is easy to assay in a normal spectrophotometer, 2) can be
expressed in bacteria, 3) can be purified using affinity chromatography, and 4) that we can
get our hands on. We have found one in a nice paper by Halliwell et al. published in 2001 in
the journal Analytical Biochemistry. Feel free to read the paper, found in the Appendix,
which describes an experiment that can be done with this system.
Overview of the Lab:
Our task this term is to take an enzyme from gene to functional protein, which we will then
analyze kinetically. The enzyme we will be studying is lactate dehydrogenase (LDH), which
interconverts lactic acid and pyruvate in the body:
Pyruvate is a metabolite of glucose, which under normal conditions (in the presence of
sufficient oxygen) enters the citric acid cycle and produces ATP (the bodys main source of
energy) and carbon dioxide. When there is insufficient oxygen, like during strenuous
exercise, this cycle cannot be completed and instead lactic acid is formed. It builds up in
muscle tissues and causes pH to fall, resulting in muscle cramps and fatigue. Eventually, it is
transported to the liver, where LDH converts it back to pyruvate, which can be used to make
glucose! We will study these processes in much greater detail later in the term.
To obtain the desired enzyme, we will first have to make multiple copies of the DNA. We
will then incorporate the DNA into a bacterial plasmid (called a vector), and then move the
plasmid (transformation) into a bacterial cell where hopefully it will be transcribed and
translated into a functional enzyme! We will then purify this enzyme out of the expression
bacteria and perform various assays on it to assess its activity.
General Lab Procedure:
We will work in pairs. While there will be no weekly write-up, there will be one final lab
research paper due from each student at the end of the term. Bear in mind that finals are also
at the end of the term, so the more work you do along the way the easier it will be for you to
write it up in the end! Take careful notes, and be sure you understand what we are doing
each week. The Halliwell et al. paper (included in the appendix) should give you a nice idea
of what is included in a research paper.
In addition, there will be pre-lab questions due at the beginning of each lab period. These
are not meant to be time consuming, in fact they are meant to ensure that you understand
what you will be doing in lab each week and thus should make the actual lab period move a
lot faster. As in organic chemistry, you will not be allowed to take your lab manual to lab
with you. You are responsible for writing out the procedures beforehand unless otherwise
noted. At the end of each lab period, you will leave your notebooks with your TA, who will
check your answers to pre-lab questions and make sure you are taking thorough notes and
that you generally understand what you are doing. Lab manuals will be available for pick-up
two days after your lab period so that you can prepare for the next week.
Lastly, get excited! This lab is not meant to stress you out, but to give you a good
introduction to the way that biochemical research is actually done. Have fun with it!
THROUGHOUT THE TERM, YOU WILL WORK WITH A LAB PARTNER.
BEFORE STARTING, SEE THE BOARD AND ASK THE TAs IF THERE HAVE
BEEN ANY MODIFICATIONS TO THE PROTOCOL.
ICE IS NICE: A LOT OF THE THINGS WE WORK WITH MUST BE KEPT ON
ICE. UNTIL YOU DO KINETICS IN WEEK 6, IT IS RECOMMENDED THAT YOU
KEEP EVERYTHING ON ICE ALL THE TIME!
A CLEAN LAB IS A HAPPY LAB. PLEASE CLEAN UP ANY SPILLS, WASH ANY
RANDOM GLASSWARE, AND MAKE THINGS TIDY BEFORE YOU LEAVE.
DO NOT USE TAP WATER FOR THE REACTIONS. USE ULTRA-PURE WATER!
DO NOT PUT TRASH IN THE GLASS DISPOSAL BOXES!
FINALLY, DO NOT THROW OUT THINGS YOU WILL NEED IN SUBSEQUENT
WEEKS!
Lab Schedule
Week One April 6th: PCR, plasmid restriction digest, phosphatase treatment, PCR
purification, PCR digest, agarose gel
Week Two April 13th: Gel purification, prepare solid and liquid bacterial media, Ligation,
transformation, plate bacteria
Week Three April 20th: Analytical DNA Analysis, Bacterial transformation, preparation
of buffers for protein prep.
**This week you will have to come in briefly (for a half hour) the day before your lab
Week Four April 27th: Purify protein
Week Five May 4th: Characterization of protein (purity, concentration, activity)
Week Six May 11th: Kinetic analysis of protein
Week Seven May 18th: Individual Experiments!
Week Eight May 25th: Individual Experiments!!
Week One
Outline
We will start out with a tiny amount of our LDH gene in pBR322 (pretend its a really rare
gene that were studying, and we were only able to get a little bit of the DNA). In order to
produce a lot of protein and be able to get any useful results, we are obviously going to need
many, many copies of the gene. Thus, this week we will amplify our tiny bit of DNA using a
common biochemical method called PCR. We will then purify our LDH gene out of the
PCR reaction mixture and digest it for next week, when we will incorporate it into our
second piece of DNA, the pET28a expression vector (shown on the next page), which will
ultimately transcribe our protein for us:
Transcription of the LDH gene is regulated by a version of the lac operon, which allows us to
effectively turn on the transcription and subsequent translation of the LDH gene. You may
already know about the lac operon, especially if you took Bio 16. If not, dont worry, it will
be explained in a later lab when we actually use it to induce expression of the LDH gene.
pET28a also contains a kanamycin resistance gene (Kan), which we will use to select cells
that successfully take up the pet28a vector. But more about that later
To prepare for the incorporation of LDH into the pet28a vector, this week we will digest
(cut) the pET28a vector using restriction enzymes HindIII and NdeI, creating an entry space
for the LDH gene. We will then the treat pET28a with alkaline phosphatase to keep the ends
from ligating (sticking) back together. We will then be digesting the purified PCR product
just like we digested the pET28a. Then, well run both DNA fragment mixtures on a DNA
Agarose gel to isolate the two fragments that we want. The above restriction maps are
important to lab this whole term and will be important in your paper. This figure, and
all others, are in the Appendix and can be torn out and pasted into your lab notebook,
should you desire.
Additionally, well make up stock solutions both for our transformation and for our agar
plates, on which well grow up our transformed cells, but more on transformation next week!
New Techniques:
PCR
PCR is a way to make multiple copies of a section of DNA off of a single double-stranded
template.
How it works:
When DNA is heated above a certain temperature (annealing temperature), hydrogen bonds
that hold complementary strands together break, and the DNA separates into two single
strands. When the temperature is lowered again, the hydrogen bonds reform. So, say we put
a little bit of our double stranded DNA to be replicated in solution with a lot of free
deoxyribonucleotides (the building blocks of DNA), some DNA polymerase (the enzyme that
catalyzes DNA replication), and a lot of short little strands of DNA that are complementary
to the DNA template strand called primers, or oligos. There are two kinds of primers: one
complementary to the DNA coding strand downstream of the gene, and one complementary
to the anticoding strand upstream of the gene.
So imagine this: we take this mixture and we heat it up to break the hydrogen bonds between
our strands of DNA template. Then we let the temperature cool. Since there are so many
more primers in solution than template strands, chances are our primers are going to end up
forming hydrogen bonds to the template before the template strands have a chance to anneal
together again. Now, the DNA polymerase can come in and, starting from these primers,
replicate the template DNA strands 5 to 3. Now, there are two double stranded copies of
the DNA encoding for the LDH gene. This process can be repeated multiple times, and each
time we melt one DNA double strand, we get two double stranded copies back (see figure
left). If we repeat this a bunch of times, well end up with a lot of copies of the gene!
Check out: http://www.youtube.com/watch?v=_YgXcJ4n-kQ
To prevent this self-ligation from happening to our plasmid (since, remember, we want to
stick our gene in there!) we use something called Calf Intestinal alkaline Phosphatase (CIP).
This catalyzes the removal of the 5 phosphate group from the cut plasmid, preventing the
DNA from joining back together.
Procedures:
1. PCR protocol for the LDH gene
We already have our two primers for the replication of the LDH gene in the pBR322 vector.
These are:
Primer I - HindIII Oligonucleotide
5`GAGGCCCTTTCGTCTTCAA3`
Normally, PCR oligonucleotides (oligos for short) would contain restriction enzyme sites that
need to be introduced in order to clone the DNA into a new vector. In our case, these sites
are already present at the ends of the LDH gene in the pBR322 vector, so were just using
these sites as the places where the oligos can anneal for the PCR. If we didnt already have
restriction sites on either side of our gene and were planning on inserting it into another
plasmid, we could use oligos that contain the restriction sites at one end which would not be
complementary to the plasmid DNA, along with a part that anneals to the plasmid. Thus,
when we ran the PCR, we would end up with multiple copies of the gene with newly created
restriction sites on either side, ready to be inserted into a vector cut by the same enzymes.
In the special thin-walled Eppendorf tubes, EACH GROUP should make up the PCR
mixture:
Ingredient
Order of Addition
40 l H2O
5l 10X PFU Buffer
1l DNA temp (100 ng/l)
1l Primer I (10 M)
1l Primer II (10 M)
1 l dNTP (10 mM each)
1l PFU TURBO Polymerase (2.5 U/l)
_________________________________
50l TOTAL
1. H2O
2. buffer
3. DNA template
4. DNA primer I
5. DNA primer II
6. dNTPs
7. Add enzyme immediately before starting!
Bring this tube over to the PCR machine as quickly as possible because all groups will start
PCR at the same time.
As we have only one PCR machine, wait for all the groups to be ready!
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METHOD
Cycle
Temp
Time
1X
25X
95 C
95 C
55 C
72 C
72 C
4C
2 min.
30 sec.
30 sec.
60 sec.
5 min.
24 hrs.
1X
Hold
5`A^AGCTT..3`
3`T TCGA^A.5`
5`CA^TATG...3`
3`GT AT^AC..5`
1. 6 l water
2. 2 l 10X CutSmart buffer
3. 10 l DNA
4. 1 l Nde1 restriction enzyme
5. 1 l Hind III restriction enzyme
Total reaction volume = 20 l
Incubate this reaction mixture for one hour at 37 degrees Celsius.
3. CIP treatment
Add 1l (1 unit) of CIP to your 20 l digest of the pET-28a plasmid. Let the enzymes work
for at least 15 minutes at 37 degrees Celsius.
4. PCR Purification
For the purification of the PCR product, we are going to use a DNA Clean & Concentrator
kit. We will be using a lot of kits like this throughout the lab they make purifications really
easy and if you decide to do more with biochemistry youll be using them a lot too. The
protocol that comes with the kit is at the end of this lab section. Since it is just as important to
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learn how to follow manufacturers procedures, you may photocopy this page and tape it in
your lab notebook. Follow this procedure to purify your PCR reaction.
comb with the proper number of lanes (use tape over two lanes if necessary the TA will let
you know how many you will need). Add 5 l of the SYBR Safe DNA gel stain to your gel
flask and mix well.
Pouring the Gel
Remove the lid from the gel box and take out the rectangular gel tray. Turn the tray
sideways and put it onto the platform in the gel box, with the open ends of the tray up against
the sides of the gel box (creating a seal). Make sure the tray is pressed down flat on the
platform. Pour your 50 ml of gel material (with added stain!) into the gel tray. Insert the
comb into the slots on the gel tray with the thicker and/or slotted side down. Let the gel
harden (this should take about 30 minutes).
Preparing the Samples
Mix each sample with loading buffer. The loading buffer is supplied in 6x concentration, so
mix one part loading buffer to five parts sample in order to get the proper running
concentration. The buffer contains glycerol so it will be very thick, but this is necessary in
order to weigh the sample down so that it will stay in the well and not disperse into the
running buffer. Carefully load your digested samples into two wide lanes, separated by
the DNA ladder lane. Add 5 L DNA ladder. If the sample starts to flow out of the well,
you have added enough! There is no need to add running buffer to the DNA ladder it
already contains it.
Running the Gel
Once the gel has hardened, carefully pull the gel tray out of the box, rotate 90 degrees, and
place it back on the platform with the comb closest to the black electrode (the cathode). Pour
1X TAE buffer carefully into the sides of the box until it covers the gel by about a quarter
inch (or 6 mm) Tap the comb to loosen it and gently pull it out of the gel. Make sure you
have the gel box near the voltage supply you are going to use, as you will not be able to
move the box again once the sample is loaded. Load the samples into the wells using the
flexible gel-loading pipette tips, being careful not to puncture or rip the gel. Note which
sample is in which well in your notebook. Place the lid back on the box and make sure you
attach the correct cord to the correct electrode. Make sure the voltage source is on run
at 150 Volts. Check the gel every so often to make sure the blue bands are running and that
the voltage source is still on. It should take 30 to 45 minutes to run sufficiently. Check with
your TA before stopping your run.
13
This is the DNA ladder which will allow you to figure out how large your bands are on
the agarose gel.
14
Clean up the buffer from your gel box carefully remember it has ethidium bromide in it
from the gel wearing gloves and pouring the used buffer in the designated receptacle
(probably in the hood). Label your samples and freeze them for next week.
15
Determine the
3. If you want to make 1 mL of a reaction mixture, what volume (in l) of the following
reagents would you add? 20x enzyme; 10x buffer; 50x NaCl; water.
4.
Why do we only CIP treat the plasmid and not both the plasmid and the PCR
product?
5. How much agarose should you add to 50 ml of TAE buffer to make a 1% solution?
6. If you forget which way to hook up your gel box, what logic can you use to figure out the
correct direction to run the gel knowing red electrode is positive and the black is negative?
7. What are the expected band sizes from an Nde1/HindIII digestion of: pBR322; pBR322LDH; pET28a; pET28a-LDH, the LDH PCR product?
8. How much kanamycin do you need to make the necessary volume of Kan stock?
16
17
Week Two
Outline:
Make LB media
Finish gel purification of DNA fragments
Set up ligation reactions
Finish preparing LB media and LB+ agar media
Transform ligations into competent cells for replication
Pour plates for bacterial growth
Once we have our new plasmid, we will insert it into E.coli bacterial cells, where the plasmid
will be replicated and amplified for us. You might be wondering (or you should be) why we
dont just have the E. coli simply replicate the protein for us. The answer is simple: it cant.
18
In week one, we briefly introduced that the LDH gene is preceded by the lac operon when
inserted into the pET28a vector. Next week, when we transform our plasmid into the actual
expression cell, well go over the lac operon briefly. For now, suffice to say that the E. coli
polymerase is not the right one for the lac operon, and thus cannot induce transcription. So
E. coli cant produce our LDH enzyme for us. What E. coli IS really good at is replicating.
Thus, we can use it to make a LOT of copies of LDH so that when we transform into the
expression cells, well have LOTS of chances for successful transformations and will
maximize the amount of protein we make!
The process of putting a DNA plasmid into a cell is called transformation. How will we
know which cells actually successfully pick up the plasmid? Since the pet28a vector
contains a gene for kanamycin resistance, only the cells that have successfully picked up the
plasmid will survive in the presence of kanamycin. Thus, we will be adding kanamycin to
LB agar media that we prepared last week so we can select for cells containing our vector.
New Techniques:
Transformation of a plasmid into competent cells
The plasma membranes of cells are fairly impermeable to most molecules, especially large
polar or charged molecules, as the plasma membrane is largely composed of hydrophobic
lipids. Thus, in order to get the DNA plasmid (a large, negatively charged molecule) into the
cell, we have to weaken the membrane a little. This has been done by the company through
the addition of chemicals. Additionally, we will heat shock the cells and the cool them down
again.
Youll notice that in the procedure for the transformation it tells you to heat shock the cells
for exactly 30 seconds. This is really important, as too long in the heat could damage the
cells and not long enough may not give the cells time for the membrane to actually take up
the plasmid. So time carefully.
Spreading Plates
A disposable bent rod serves as a plate spreader for our purposes. Pipette the transformed
cells onto the plate, and spin the plate, holding the rod steady, so that the cell mixture gets
spread evenly over the plate. The TA will demonstrate this once in lab. Place the lid on,
label the plate, and place the plates upside-down in the incubator. We will incubate them for
one night to allow the colonies to form, then halt their growth at 4 degrees Celsius until next
week.
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Procedure:
1. Finishing the LB media and LB+ agar media
ALL BUT ONE GROUP WILL DO THIS: Empty your LB ingredients from last
week into a 1L flask and add 1 liter of H2O. Shake until all solids are mostly dissolved.
ONLY ONE GROUP WILL DO THIS: Add 15 grams bacto-agar to enough LB for 1
liter and dissolve in 1 liter of water. Shake until all solids are somewhat dissolved. We
will use this for pouring plates later.
BOTH OF THE ABOVE GROUPS WILL DO THIS: Cover all the flasks with foil,
put a piece of autoclave tape on them (label with your initials), and autoclave for 30
minutes at 15 psi. Once the media solutions have cooled to about 50 Celsius, add the
appropriate amount of stock kanamycin solution, which is at 10 mg/mL in water, to a
working solution of 25 g/mL in the media.
FOR THE GROUP WITH AGAR IN THE LB: Quickly (before it hardens) pour the
agar-containing solution into petri dishes. Allow everyone in lab to pour at least one
plate.
All other groups will autoclave their media in a one liter bottle. Pour you LB into the
bottle and add one liter of water.
2. Gel Extraction of fragments
While the media is in the autoclave, follow the directions for the kit provided (you may
photocopy the directions and put them in your notebook) to purify the DNA segments.
Note that you must first determine the mass of each band. Elute both pieces of DNA
with 30 uL of water in the final step.
3. Ligation Reactions
The DNA ligase comes in a ligase buffer, called T4 buffer, which contains 50 mM TrisHCl, 10mM MgCl2, 10mM DTT, 1 mM ATP, and 25g/mL BSA. The manufacturers
recommend a DNA concentration of 0.1 to 1 M 5 termini. Thus, we will set up our
ligation reactions as follows:
Reaction
2 l CIPd plasmid
2 l PCRd LDH gene
1 l T4 ligase
2 l 10x ligase buffer
13 l H2O
Control I
Control II (+ )
2 l CIPd plasmid
----------1 l T4 ligase
2 l 10x ligase buffer
15 l H2O
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4. Transformation
1. Place the required number of sterile 1.5 ml microcentrifuge tubes on wet ice.
2. Gently mix cells with the pipette tip and aliquot 40 l into each microcentrifuge tube.
3. Pipette 3 l of each ligation reaction directly into the competent cells and mix by
tapping gently. Do not mix by pipetting up and down. Store the remaining ligation
reaction at -20C.
4. Incubate the vial on ice for 20 minutes.
5. Heat-shock for exactly 30 seconds at 42C. Do not mix or shake.
6. Remove vial from the 42C bath and place on ice for 2 minutes.
7. Add 500 l of SOC medium to each vial and transfer transformations to a culture
tube. Push the cap down to the first stop.
8. Shake the reactions at 37C for 40 min at 225 rpm in a shaking incubator.
9. Spread 200 l from each transformation vial on separate, labeled LB+Kanamycin
agar plates. Incubate overnight at 37C, then store at 4C until next week (the TAs
will put the plates at 4C the next morning).
Week 2 Questions:
1) Why do you think the T4 ligase buffer contains ATP?
2) Explain the control used in the ligation reactions. What do we expect will (ideally)
happen in the control reaction? If you do get some colonies on the control plate, what
does this mean?
3) What other control might be a good idea to do for this experiment?
4) Why should you add the kanamycin after autoclaving?
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Week Three
Outline:
THE DAY BEFORE LAB
Preparation of overnight cultures
THE DAY OF LAB
Minipreps (DNA extraction)
Analytical Digest and Gel
Transform the plasmid into the expression cells
Prepare the buffer for the protein prep
Purpose and Theory:
This is a VERY full week, so make sure you come into lab knowing what you have to get
done and do it efficiently! The good news is that most of the procedures you will be doing
this week are things that you have already done, so you should be pretty familiar with them.
Last week we attempted to ligate our LDH gene into an expression vector. As you know,
there are lots of things that can go wrong with such DNA manipulations. Our goal this week
is to analyze our ligation products and hopefully identify the complete expression construct.
In order to do this, we will select bacterial colonies from our transformation plates. Each
colony is descended from a single bacteria that survived the transformation process and has
acquired kanamycin resistance. Now, we have to figure out which colonies acquired the
desired pet28a-LDH construct. To do this, well break open the cells, collect the DNA, and
perform an analytical digest and gel. Youve done both of these before, so this part should
be easy!
Once we find the correct construct of the plasmid, well transform it into the expression
bacterial cell line, BL21(DE3). This particular strain of E. coli does have the right
polymerase for the LDH gene. However, those of you who are familiar with the lac operon
will know that induction of the lac operon is not as simple as having the right polymerase.
The lac operon, in its normal biological context, controls the transcription of -galactosidase,
galactoside permease, and thiogalactoside transacetylase. These three facilitate the
metabolism of lactose. However, the cell does not want to waste energy on these when
lactose is not present. Thus, the synthesis of these genes is regulated by two control sites that
are 5 to the genes: the operator and the promoter. The promoter is the location on the DNA
where the polymerase binds. It would bind all the time if it could, but it doesnt because of
the second regulatory site, the operator. This site binds the repressor, which is encoded for
elsewhere on the DNA. In its natural state, the repressor blocks the binding of the RNA
polymerase. However, the repressor can be induced to let go by the presence of lactose,
which binds in an allosteric position on the repressor protein and changes its conformation.
Once the repressor is off of the DNA, the polymerase is able to bind and transcription and
translation of the proteins can occur:
23
In our system, these three lactose metabolism genes are replaced by the LDH gene, but the
regulation of the transcription of this protein works the same. Basically, we have to induce
the repressor to change shape and fall off. We technically could use lactose, but lactose
would be broken down by the BL21(DE3). So, we use IPTG, which looks a lot like lactose,
and even binds to the repressor protein and causes it to fall off, but cannot be metabolized so
we can repeatedly transcribe the LDH gene and make lots of our enzyme!! Since we didnt
want to make you guys come in extra days again (even though we know you just cant get
enough of lab) well induce the expression cells for you before next week, but you should
still understand how its done.
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Procedure:
THE DAY BEFORE LAB
1. Preparation of Overnight Cultures
EACH GROUP: Retrieve your plates from the refrigerator. If you have six or more
colonies on your LB kanamycin plate, pick six of them. To pick a colony, touch it with a
yellow pipette tip on a micropipet and eject the tip into 2 mL of your LB-Kanamycin media
(that you finished making last week) in a plastic 14 mL falcon tube. Place these in the
shaker, which should be set at 37 degrees Celsius. These should grow into saturated cultures
overnight.
THE DAY OF LAB
2. Minipreps (DNA Extraction)
Next, we will extract the DNA from the six cultures grown overnight.
See the end of this section for the protocol, which you may put into your notebook.
3. Analytical Digest
The easiest way to make sure you have the construct is to take some of your DNA (Dont use
all of it! You still need some to transform into the expression cells to make LDH!), digest it,
and run it on a gel to see if you get the fragments you expect. By now, you guys should be
experts at running digestions, so go for it! You are using the same enzymes as in week one,
so all the buffers and concentrations are the same. A trick here is to make up in one
Eppendorf tube enough buffer, enzymes, and water to do the eight reactions (You should
always make up more than enough of this type of stock, so make enough for 10 digests).
Then aliquot out the proper volume of the digest mixture into the tubes and add the DNA.
This saves you a lot of pipetting.
4. Analytical Gel
You should also be experts at running DNA gels by now! Prepare a gel just as before, and
load your samples with the right amount of 6x loading buffer. Since they have 12 lanes total,
load the DNA ladder in the leftmost lane, and your six samples in the other lanes. Make sure
you keep your tubes straight at this point. If you get the right plasmid but confuse the tubes,
you will not make any protein! When your gel is run far enough, take a picture and see if you
got your plasmid.
25
5. Transformation
Once you know which of your cultures has the correct form of the plasmid, you can
transform the plasmid into the expression bacteria, called BL21(DE3). Before next weeks
lab, the TAs will pick one colony from your plate, inoculate a ~1 liter culture of LBkanamycin, and then induce protein expression with IPTG.
1. Pipette 5 l of each plasmid prep directly into your 100 l aliquot of competent cells and
mix by tapping gently. Do not mix by pipetting up and down. Incubate the vial on ice for
30 minutes.
2. Heat-shock for exactly 30 seconds at 42C. Do not mix or shake.
3. Place cells on ice for 5 minutes
4. Add 150 uL room temperature SOC
5. Shake the reactions at 37C for 45 minutes to 1 hour at 225 rpm in a shaking incubator.
Spread the transformed cells on one of your leftover LB-Kanamycin plates from last week
and place in the 37 degree incubator for overnight growth.
26
Week 3 Questions:
1. Is it possible to find a colony on your LB kanamycin plate that does not contain the
LDH gene? What could have happened to allow this colony to grow?
2. What fragments are you looking for on your analytical gel that would ensure you got
the proper construct?
3. Work out how much of the buffer, enzymes, and water you will need to add to one
Eppendorf tube in order to make a 10-reaction master mix, as suggested in the
procedure.
4. Use the Henderson-Hasselbalch equation to figure out how to make 1 liter of the
following buffer: 50 mM sodium phosphate, 300 mM NaCl, at pH 8.0. The relevant
pK here is 6.82. (sodium phosphate monobasic NaH2PO4, FW 119.98 g/mol), sodium
phosphate dibasic (Na2HPO4, FW 141.96 g/mol).
5. Why do you use phosphate buffer at pH 8.0 when the pK of phosphate is 6.8?
27
28
Week Four
Outline:
29
Affinity chromatography
Remember way back in the first lab when we discussed PCR and how it can be used to
introduce a poly-histidine tag (which we actually already had)? Well, were finally going to
get to use it.
The active groups on the Ni-NTA resin that is in your column are shown above. The NTA
(nitrilotriacetic acid) chelates the Ni2+ in four of its six preferred positions. The other two
sites are then available to interact with two histidine residues from your protein tag,
completing an octahedral geometry, as shown above. This means that the tagged protein
binds while other proteins will flow through the column, unless they just happen to have a
series of available histidines, which is very rare (but this does happen, so be careful if you
ever use this in the future!). We then use imidazole to compete off the histidines, thereby
eluting the protein.
The quick and dirty Bradford Assay
Well talk about how you actually use a Bradford assay to find the concentration of
your protein next week, but for this week, were just going to use it to locate the protein, not
to quantify how much there is.
Dialysis
In order to remove the high concentration of imidazole from our pure protein, we will use
dialysis to exchange the buffer.
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Procedure:
1. Separation of the protein from the cell
The TAs will have inoculated and induced your ~1 liter of LB-Kan with a colony from your
latest transformation plate. They have also taken the cells and spun them down and then
resuspended them in 50 mL of 10 mM imidazole protein buffer that you prepared last week.
Take these resuspended cells and place in a small beaker with a stir bar for sonication. The
cells are broken up by sonication, which uses ultrasonic waves to break open the bacteria.
The process takes 8 minutes, consisting of 4 cycles of 1 minute on, one minute off. The off
cycles allow the cells to cool between sonications.
Save 50 l of your sonicated cells for gel analysis next week!
After the cells are broken, the insoluble material is separated from the soluble material
(which includes your LDH) by high speed centrifugation. Well do this in the lab in Steele.
Two groups should spin at a time, and the tubes should be placed symmetrically in the
centrifuge. Aliquot your ~60 ml of broken cells into two centrifuge tubes, make sure they are
balanced (that is, both must weigh the same it may take a few transfers to get this right, but
it is important!), and put the tubes opposite each other in the small rotor. They should spin
for 15 minutes at the maximum speed (between 12,000 and 15,000 rpm) allowed by the
machine (indicated on the control knob).
2. Column Purification of the LDH protein
While the spin is going, pour your column. Close the valve at the bottom of the column and
pipette in 5 ml of the Ni-NTA matrix slurry (10 ml of this stuff costs $65, so please be
careful). Then open the valve and allow it to settle (make sure you have a beaker or tube
under the spout to collect the flow through). Dont let the liquid level get all the way down to
the top of the column, though. The column must not run dry!
When the material is settled, put the plunger in the column (we will demonstrate how to do
this the plunger must go down close to the top of the Ni-NTA column level but must not
disturb it! Start to equilibrate the column with 50 ml of the 10 mM imidazole buffer, B2.
You can pump at 3 ml/minute. When enough buffer has passed over the column (measure
this by collecting the flow-through with an orange flask or anything else with mL markings
on it), stop the pump and close the column valve.
By this time, your cells should be done centrifuging. The supernatant should look clear.
Pour the supernatant off carefully into a clean beaker or a 50 mL cylindrical centrifuge tube
on ice. MAKE SURE YOU KEEP YOUR PROTEIN AND BUFFERS ON ICE AT ALL
TIMES! Save 50 ul of this soluble protein sample for gel analysis next week! Place the
pump end into your supernatant and start loading it onto the column at 3 mL/minute. From
the time you start loading the protein on until you are finished with the column, you should
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collect the flow-through. For the first three wash steps, you may collect all of the flow
through from each wash in a separate, single tube and save it, whereas for the elution step we
will collect the flow-through in smaller fractions.
Once the protein is loaded (and the flow-through from the loading step labeled and saved),
wash the column with 30 mL of the 10 mM imidazole buffer, B2 (measuring the amount
gone through from the marks in the collection tube) at 3 mL/minute. Label and save the
collected flow-through.
Next, wash with 30 mL of the 40 mM imidazole buffer, B3 at 3 mL/minute. Label and
save the collected flow-through.
Next is the elution step! Set up the fraction collector with 15 tubes (just in case you will
probably only use 10). Set the pump/collector to collect ten 2-ml fractions and to pump at 1
ml/minute. Elute with 20 ml of 250 mM imidazole buffer, B4. Make sure to press both the
timer and start buttons on the pump, or the fraction collector will not work.
3. Preparation of Gel Samples for Next Week
Take 50 l of each fraction in small, labeled Eppendorf tubes to test on a gel next week.
4. Quick Bradford Assay
Well do a more quantitative Bradford Assay next week after dialysis (well make the
standard BSA curve and all that other fun stuff as described above), but for now we just want
to figure out which fractions have protein so we can pool them. Set up 10 clear eppendorf
tubes in a rack. Then combine in the eppendorf tubes 40 l of your fraction and 10 l of
assay mixture. Do this for each elution fraction. Note which fractions turn blue, since the
blue color indicates the presence of protein. Well then pool these fractions and dialyze the
protein for you to use next week.
SAFETY NOTE: The Bradford reagent contains methanol and concentrated
phosphoric acid! Wear gloves and goggles.
5. Dialysis
Pool the fractions that contain protein (not the blue Bradford tubes, the ones from the
column) and transfer this into dialysis tubing. Your samples will dialyze overnight and then
the TAs will aliquot them and freeze all except one for the next two weeks!
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Week 4 Questions:
1. Why does increasing the concentration of the imidazole make the protein come off of the
column?
2. What looks strange in the figure shown above of the Ni-NTA taken from the Qiagen
handbook?
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Week Five
Outline:
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Procedure:
1. SDS Polyacrylamide Gel
Fortunately for you all, the system we use in Chem41 lab is almost totally automated. Add 5x
Laemmli Buffer (blue) to each of your saved samples from last week and load a small
amount of each onto the gel, add a protein ladder sample, and let it run. After an hour or so,
youll have a nice gel for your notebook (and lab write-up!).
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3. Activity Assay
The assay buffer for the activity assay is: 20 mM TRIS-HCl, 50 mM KCl, pH 7.0. The TAs
will provide this for you!
Your enzyme should be (we hope so!) very concentrated, so we will dilute it in assay buffer.
Use serial dilutions to get to a reasonable reaction rate. (serial dilutions are 1:10, 1:100, etc.
Make one dilution, then dilute the dilution by 10, etc.) A serial dilution is a nice way to
accurately dilute your enzyme. Add 90 l of assay buffer to 3-4 tubes. Then add 10 l of
your enzyme to the first tube and mix. Remove 10 l of this and add it to the second tube,
mix, and repeat. Each tube will be a 1:10 dilution of the prior one.
For example, start by diluting your enzyme 10x by adding 10 l of enzyme to 90 l of assay
buffer.
Then, try running a reaction. To do this, mix in a cuvette:
900 l assay buffer (at room temperature!!)
33 l NADH
33 l pyruvate
(We will provide you with the NADH as 6.6 mM in assay buffer and Na-pyruvate as 30 mM
in assay buffer)
To this, add 33 l diluted enzyme, mix quickly, and immediately observe absorbance at 340
nm (see the UV spectrum of NADH on the following page note this data is so old (but still
good) that NADH was not even called NADH! You will probably want to bring your ice
bucket over to the spectrophotometer so you can get the cuvette in quickly. It should start
around A340 of 1.4 (you have to be fast to get this!) and decrease rapidly over a minute or two
to a baseline level, where all the NADH is oxidized. We are looking for a linear change in
absorbance over one minute, ideally - 0.1 - 0.2 absorbance unit per minute. Run the reaction
for 2-3 minutes. If your reaction is too fast (and it almost certainly will be), using a serial
dilution, dilute the enzyme 1:10 and try again, etc. When you find a reasonable enzyme
concentration, you can dilute it 1:2 serially instead of 1:10 to narrow in on an ideal
concentration, Make sure you keep track of the dilutions so you know the amount of enzyme
in your assay and how to dilute it to the ideal concentration as we will use this dilution over
the next two weeks in doing experiments on our LDH!
Once you have found your ideal dilution and get a nice looking curve, you can calculate the
activity of your enzyme using the following formula:
Where 6.22 ml cm-1 mol-1 is the extinction coefficient of NADH (in the correct units).
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Units are a somewhat arbitrary unit describing enzyme efficiency. As commercial enzymes
have different activities and purities, they are often sold in Units, and then the Unit is defined
in some way. So for a restriction enzyme like HindIII, one might buy 50,000 U at 20,000
U/ml. The HindIII Unit is defined as the amount of enzyme required to digest 1 g of DNA
in 1 hour at 37C in a total reaction volume of 50 l.
For LDH, the commercially available enzyme comes in batches of 25,000 Units, at around
250 U/mg. The LDH Unit is defined as the amount of enzyme needed to oxidize one
micromole of NADH per minute at 25C, pH 7.3.
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Week 5 Questions:
1) (You cant do this one ahead of time) Based on your calculated concentration from the
Bradford assay, figure out how many total units you have of your enzyme. We can buy LDH
for about $170 per 25,000 Units. How much is your enzyme prep worth?? (youll have to
wait until you actually find the concentration in lab, obviously, to finish this, so just figure
out how youll do this problem ahead of time so it will be easy to do in lab!)
2) What is the expected molecular weight of your purified LDH protein?
3) What are some problems inherent in using a BSA standard curve with a Bradford assay to
quantitate LDH? Propose a better protein assay for LDH.
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Week Six
Outline:
Michaelis-Menton Kinetics!
Purpose and Theory:
This week our goal is to determine the Michaelis-Menten kinetic parameters for our purified
enzyme. As you remember from class, a plot of vo vs. [S] for various [S] will give us a plot
that on the left; and 1/vo vs. 1/[S] (a Lineweaver-Burke plot), like that in the middle; and vo
vs vo/[S] (Eadie-Hofstee), like the one on the right.
New Techniques:
Determining a Michaelis-Menton Constant for an Enzyme
First of all, you need a good working dilution for your enzyme that allows for a clear
determination. We did this last week. Then, we need a substrate to vary. It wouldnt help us
much to vary NADH, since we are measuring its absorbance and if we start diluting it, we
wont get any useful readings. Therefore, well dilute pyruvate in serial dilutions instead.
Well then perform activity assays like we did last week.
Use the extinction coefficient for NADH to determine the rate of NADH oxidation for each
case. Remember A = cl, where c = concentration (starts at 30 mM) and l = path length (1
cm). Plot this vo (in terms of d[pyruvate]/dt) versus [S] or 1/[S]. You can use a positive value
for all vos, but watch out for units! Calculate the Km and Vmax values for your enzyme. Do
the two plots agree? You can also calculate kcat, which equals Vmax/[E]t.
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Procedure:
1) Dilute your enzyme to the working concentration you determined last week. You will
need about 1 ml of your diluted enzyme. Use the assay buffer to dilute it.
2)
3)
4)
Make a 1:2 serial dilution of pyruvate (our varying [S]). Start with the 30 mM pyruvate,
and do 8 dilutions by adding 100 l of pyruvate to 100 ul of assay buffer, mixing, and
then going on to the next tube. Start with 200 ul pyruvate. You should have 9 tubes in the
end, with 100 l in each (except 200 l in the last):
Do an activity assay, just like last week, for each dilution. Do each measurement in
duplicate to be thorough!
Calculate the Km and Vmax values for your enzyme. Do the plots agree? Also calculate
kcat:
Week 6 Questions:
1. How you will dilute your enzyme to the correct working concentration
2. Fill in the following table for your pyruvate serial dilutions:
Tube
[pyruvate] mM
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temperature
pH
ionic strength
type of buffer
Plan with your lab partner ahead of time really do put a bit of thought into it since this is
pretty much the conclusion of your lab experience! Please OK your experiment with the TA
of the Professor, especially if you want to do something strange. Have fun! You are doing
REAL science now!
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Appendix 1
Halliwell CM, Morgan G, Ou CP, Cass AE. (2001) Introduction of a (poly)histidine tag in Llactate dehydrogenase produces a mixture of active and inactive molecules. Anal Biochem.
15;295(2):257-61.
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