D. M. Dean and J. A. Wilder
The Frankenplasmid Lab
(from our course Laboratory Manual)
(File S1)
In class, you have been hearing about how molecular biologists use techniques such
as restriction mapping and the polymerase chain reaction (PCR) to characterize a
DNA sequence. You have also been learning that restriction fragments from different
DNA molecules can be combined in solution and ligated together (assuming that these
fragments have compatible ends), resulting in various species of hybrid, or
“recombinant” DNA. This ligated DNA can then be transformed into bacteria, and the
cells plated on agar-based media. Media additives such as antibiotics or X-gal can
select for the presence of certain genes (and therefore certain fragments) within the
transformed plasmid. Transformation is a rare event, and so a single colony on
selective media is in all likelihood derived from a single cell that was transformed
with one species of recombinant DNA. This single colony can then be expanded in
liquid media, allowing a researcher to replicate a specific recombinant DNA molecule
in high quantities for further use or analysis.
The goals of this lab are to generate your own unique recombinant plasmid, replicate
it in bacteria, isolate it, and determine the orientation of the DNA fragments that it is
composed of, using restriction digests and PCR. Here is a more specific outline of
what will be done and when:
Week 1
Day 1
-turn in Prelaboratory Exercise 4-1 at the start of lab
-HindIII and EcoRI digests of pUC-Kan and pBR322
-separate the resultant restriction fragments by size using agarose gel electrophoresis
-ligate pUC-Kan and pBR322 restriction fragments together
-transform ligation and control plasmids into E. coli
-plate transformed cells on selective media
Day 3
-transfer one colony containing recombinant plasmid from your plates to liquid media
Week 2
-isolate your recombinant plasmid from the liquid culture
-HindIII and BamHI digests of recombinant plasmid
-PCR of recombinant plasmid
Week 3
-turn in Prelaboratory Exercise 4-3 at the start of lab
-agarose gel electrophoresis of HindIII digest, BamHI digest, and PCR products
1
D. M. Dean and J. A. Wilder
2
Materials/Methods
E. coli
For transformation, we will use the strain DH5, which has the following genotype:
supE44, (lacIZYA-argF)U169, (80dlac(lacZ)  M15), recA1, endA1, hsdR17 (rK-, mK+),
deoR, thi-1, glnV44, gyrA96, relA1
Most of these mutations cause losses of function in general housekeeping genes. Their
cumulative effect weakens the DH5 strain. As a result, DH5 cells do not survive well
outside of our culture conditions and are not harmful to humans.
But these mutations are also useful in three more specific ways. First, the recA1 mutation
prevents DNA recombination from occurring, so a transformed plasmid will remain
extrachromosomal and stable. Second, the lacZ mutation ( = a deletion in the listed gene)
means that DH5 does not have a functional lacZ gene, one of the three genes encoded by the
lac operon. The LacZ enzyme is normally involved in lactose metabolism, but in the presence
of an alternative substrate, 5-Bromo-4-chloro-3-indolyl-D-galactoside (X-gal), it will produce
a blue product. Third, the lacI mutation means that the transcriptional repressor for the lac
operon is produced at low levels, which will allow the lac operon to be expressed at high
levels. Taken together, we expect that untransformed DH5 will not convert X-gal and as a
result its colonies will remain white, but if these same cells are transformed with a plasmid
containing the lacZ gene, they will produce blue colonies (see Growth media below).
Finally, it is important to remember that DH5 cells are sensitive to the antibiotics
kanamycin, tetracycline, and ampicillin. This will be useful for determining the contents of
our recombinant plasmids on a genetic level.
Growth media
LB
This is standard E. coli growth media. LB, or “Luria broth”, is named after the original maker
of the recipe (Maniatis et al., 1987). It can be used in liquid form to produce large batches of
cells, or if agar is dissolved in it, the media forms a solid gel matrix, allowing the researcher
to isolate individual colonies that each descended from a single cell.
kanamycin, tetracycline, ampicillin
These are standard antibiotics used for microbiology. DH5 is sensitive to all three agents.
The following alleles, if present on a plasmid, will impart antibiotic resistance: kanR
(resistance to kanamycin), tetR (resistance to tetracycline), and bla (a mutant allele in a gene
encoding -lactamase which enables Amp resistance). By having kanR on one plasmid and
tetR on the other, and growing transformed cells on media containing kanamycin and
tetracycline, we will ensure that any colonies we isolate will contain a recombinant plasmid.
Note that, for simplicity, we will call the antibiotic resistance alleles Kan, Tet, and Amp throughout
this lab.
X-gal
D. M. Dean and J. A. Wilder
3
X-gal allows for a different type of selection than antibiotics. It does not significantly affect
bacterial growth when it is in the media, but rather is an alternative substrate for the LacZ
enzyme. LacZ activity converts X-gal into a blue product, but a colony will remain white if it
does not possess LacZ function. The lacZ gene has been deleted in the DH5 strain, so the
presence of a functional lacZ gene in a transformed plasmid is easily scored.
Plasmids
We will start our experiment with the plasmids pUC-Kan and pBR322. After
restriction digesting these vectors, you will determine the orientation of the pUC-Kan
sequence (see below for a further explanation of restriction mapping), combine the two
digests in solution, and ligate them together to generate recombinant DNA. Below is
some background about the plasmids that we will use.
pUC-Kan
The instructors synthesized pUC-Kan out of two previously existing plasmids, pUC19
and pACYC177. pUC19 is often poorly annotated online, so its essential features are
given here. It is a 2686 bp vector containing a lacZ gene. The 5’ end of the gene is at
nucleotide 469 of the published sequence, the 3’ end is at 146, and this directionality is
notated “469-146”. pUC19 also contains an origin of replication (1455-867), which
allows autonomous replication independent of the chromosome, and the Amp gene
(2486-1626). To make pUC-Kan, we first cut pUC19 with the enzyme ZraI to linearize
the vector.
pACYC177 is well-annotated online, so you will determine the location of its essential
features in Prelaboratory Exercise 4-1. To synthesize pUC-Kan, pACYC177 was
digested with AfeI. A fragment from pACYC177, containing the Kan gene, was ligated
into the ZraI site of pUC19 in either of the two possible directions. We will call the
resultant vectors pUC-Kan 1 and pUC-Kan 2.
On Week 1, you will be given one of these two pUC-Kan vectors, but not told which
direction the Kan fragment is in. One purpose of the Prelaboratory Exercise 4-1 is to
predict the two possible restriction maps. You will then be able to match one of these
predictions to a restriction digest that you will do in class. This will determine the
pUC-Kan sequence that you are starting with, allowing you to come up with further
predictions as you analyze your recombinant plasmid during subsequent weeks.
pBR322
This plasmid contains an origin of replication, the Amp gene, and the Tet gene. pBR322
is well-annotated online, so you will determine the location of these features in
Prelaboratory Exercise 4-1.
Restriction mapping
Recall that bacteria, as an immune response of sorts, produce restriction enzymes to
break down foreign DNA at specific sequences, protecting their own DNA by
methylating it at vulnerable sites. Biotechnology firms have isolated and massproduced restriction enzymes, allowing us to use them in vitro to cleave DNA at
specific sites. If we splice two DNA fragments together with DNA ligase, and know
where certain restriction sites lie within each fragment, we can determine the relative
D. M. Dean and J. A. Wilder
orientations of the two fragments by restriction digesting the recombinant DNA,
separating the fragments by size on an agarose gel, and determining their sizes.
For more information about the sequences recognized by the restriction enzymes that
we will use, see the websites of biotechnology firms such as New England Biolabs
(www.neb.com).
Polymerase chain reaction (PCR)
There are certainly other methods to deduce DNA sequence other than restriction
mapping. The most direct, of course, is to sequence the DNA, but for logistical
reasons, we will not be doing this. However, to strengthen our findings from the
restriction digest, we will use the polymerase chain reaction (PCR). Recall that this
method uses a pair of short, single-stranded DNA primers that flank a stretch of
DNA to selectively amplify that sequence. You have used PCR in 102 and will be
hearing about it in more detail in Genetics lecture. If you would like an online
tutorial to brush up, here is a useful link:
http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html
Select “Amplification” in the menu to see an animated description of the technique.
As you watch the tutorial and consider the technique, make sure you understand
that the two primers must anneal to opposite strands of the DNA template, and
their 3’ ends must point at each other in order for you to be able to amplify
intervening DNA exponentially and see it on a gel. This fact, along with our
selection of primers, will help us determine the relative orientations of the fragments
in our recombinant plasmid.
We will use three PCR primers in lab: pUC1, pUC2, and Tet. pUC1 and pUC2 anneal
to pUC-Kan, running in opposite directions, and Tet anneals to pBR322. You will be
given the sequences of these primers. In Prelaboratory Exercise 4-3, you will
determine where these primers should anneal and which direction each one runs
relative to their template sequences. This will allow you to predict the sizes of PCR
products in cases where pUC-Kan and pBR322 are appropriately oriented to produce
a double-stranded product between pUC1 and Tet or between pUC2 and Tet. As
with the restriction digests, these predictions will be compared to actual results on an
agarose gel, allowing you to determine the relative orientations of the fragments in
your recombinant plasmid.
4
D. M. Dean and J. A. Wilder
5
Prelaboratory Exercise 4-1
Turn in this exercise at the start of the Week 1 lab.
The plasmids we will use require some background information, as well as some preparation on your
part to know the significance of your results.
In this exercise, you will determine the locations of various genes and restriction sites on pUC19,
pACYC177, and pBR322. This will give you the information necessary to deduce the possible
restriction maps that you could get on Weeks 1 and 3, and even help you characterize the possible PCR
products that you could get on Week 3.
1. Log onto Pubmed (www.pubmed.org). You have probably used it when writing papers to find
primary references. However, it can also be used to find many other bits of information relating
to biological research. We will use it to gather information about the plasmids used in this lab.
2. Under Search in the top left corner, move the toggle switch to Nucleotide. This switches
Pubmed to a mode of searching for nucleotide sequences. DNA sequences are archived using
accession numbers; the one for pUC19 is L09137. Type this number into the search box next to
the toggle switch and hit Search. (Although typing “pUC19” will allow you to find the same
information, there will be a lot more options to weed through, so to save some confusion, we gave you a
shortcut.)
3. You will probably see only one option for the pUC19 vector. (They call it pUC19c.) You will see
a lot of useful information, such as references, the positions of genes on the plasmid, and the size
of the entire vector. At the bottom of the page is the nucleotide sequence. Select the nucleotide
sequence and copy (ok to include margins and flanking numbers).
4. You will now need to find the locations of restriction enzyme sites on pUC19. New England
Biolabs, a well-respected manufacturer of restriction enzymes, has NEBcutter, a very useful
online tool that finds the sites for you. In a new browser window, go to www.neb.com and click
on Technical Reference at the top. The next page will show a series of free online information. In
the left column, under Favorite Tools, select NEBcutter and paste your sequence into the large
box that appears. Below the large box, under The sequence is: select Circular because you are
dealing with a plasmid. Hit Submit to find the restriction sites.
Take a moment to examine the output. The search engine attempts to predict the positions and
directionalities of genes, but do not put too much trust in this. Note that the precise location of each
restriction site is given if you move the cursor over the enzyme name. Also, you may alter which enzyme
sites are shown and how they are displayed by playing with the Display and List options at the bottom.
Keep this in mind because, although ZraI cuts pUC19 once, some enzymes may cut a vector multiple times
or may not cut it at all, and you can control which of these categories is or is not displayed.
5. Given that we cut pUC19 with ZraI to make pUC-Kan, find the location of the ZraI site
_________. What is the size of the linearized plasmid in base pairs (bp) of DNA? ___________
6. Also find the locations of any HindIII, EcoRI and BamHI sites. The reason for this will become
clear during the exercise. HindIII ____________EcoRI_____________BamHI_____________.
7. Sketch a plasmid map that shows the total length (bp), positions of any ZraI, HindIII, EcoRI,
and BamHI restriction sites relative to the origin of replication, the Amp (-lactamase) gene, and
the LacZ gene positions that were given in the Materials/Methods section of this lab. Be sure to
D. M. Dean and J. A. Wilder
6
include the directions of gene transcription. This will not be checked by your instructors, but will
serve as a resource for your lab report. On the next page is an example of a typical format for a
plasmid map.
Figure 4-1. A typical plasmid map. Lines around periphery show positions of restriction sites and arrows
show positions of genes and their directions of transcription. Note that we are going to map restriction sites
for far fewer enzymes. (Source: New England Biolabs)
8. Return to the browser window containing Pubmed. Search for the other plasmid we used to
make pUC-Kan, pACYC177 (accession number X06402). The annotation for this sequence is
better than that of pUC19. Right above the DNA sequence is a summary of the genes on the
plasmid. Write down the the total length _________, as well as the coordinates and directionality
of the origin of replication________________, the Amp gene (i.e. bla or -lactamase)
______________, and the Kan gene__________________. The latter will be especially important to
know, because the Kan gene is on the AfeI fragment that we ligated into pUC19 to generate pUCKan.
9. You can either copy the plasmid sequence and paste it into the NEBCutter or type the accession
number into the Genebank number box at the top. (You could have used the accession number
for pUC19 as well, but we avoided this the first time through in order to give you an opportunity
to observe the Pubmed output and to understand the parameters of NEBCutter more fully.) In
either case, remember to set the engine to the Circular setting. Find any AfeI_____________,
BamHI___________, EcoRI_____________and HindIII_______________sites. Briefly, how can we
ligate an AfeI fragment into a ZraI site?____________________
10. Sketch a map of pACYC177 that includes all of the features that you just located during Steps
8 and 9. This will not be checked by your instructors, but will serve as a resource for you as you
write the lab report.
11. Look up pBR322 using Pubmed (accession number J01749). Write down the total
size___________, as well as the coordinates and directionality of the Amp
gene___________________,
Tet
gene____________
and
the
origin
of
replication__________________. Using the NEB site, determine the locations of any
BamHI___________, EcoRI_____________and HindIII_______________sites.
D. M. Dean and J. A. Wilder
7
12. Sketch a map for pBR322 that includes all of the features that you just located during Step 11.
This will not be checked by your instructors, but will serve as a resource for you as you write the
lab report.
D. M. Dean and J. A. Wilder
8
Experimental procedures
Work with the same partner throughout this experimental module.
Week 1
A. Restriction digests
In this section, you will digest the pUC-Kan and pBR322 plasmids with EcoRI to give them
compatible sticky ends. The resultant fragments will later be ligated together to generate a
recombinant plasmid. In a separate reaction, you will also digest these two constructs with
HindIII to determine the orientation of the Kan gene in pUC-Kan and to assist you in
constructing your final restriction map.
1. Check your station to confirm that you have the following reagents:
In ice bucket:
pUC-Kan-1 or pUC-Kan-2
pBR322
10X concentrated enzyme reaction buffer (“EB”)
EcoRI (“E”)
HindIII (“H”)
10X concentrated T4 DNA ligase buffer (“LigB”)
2 tubes of competent E. coli cells (“CC”)
In Eppendorf tube rack:
Sterile water (“ddH2O”)
Luria broth (“LB”)
DNA molecular weight standard (“MWS”—this is shared by a group of four)
2. Write down whether you have pUC-Kan-1 or -2. You will need this information to ask us
questions and for your report.
3. Label four clean microfuge tubes 1-4 for your restriction digests. Add your initials to identify
these tubes as yours.
4. In Sections B and C, you will share a gel with the other pair of students on your side of the
bench. Each gel will need only one set of undigested controls. Therefore, coordinate with your
neighbors to make up these controls. Working with the other pair of students, label two clean
microfuge tubes A and B. Add initials to identify them as yours.
5. Add 26 L of ddH2O and 4 L of EB to each of your tubes 1-4. Add 5 L of ddH2O and 1 L of
EB to the shared tubes A and B.
6. Add 10 L of pUC-Kan to tubes 1 and 3. Add 10 L of pBR322 to tubes 2 and 4. Add only 4 L
of pUC-Kan to tube A and 4 L of pBR322 to tube B. Mix by “finger vortex” (ask for demo if you
are unclear on what this is).
D. M. Dean and J. A. Wilder
9
7. Finally, add 2 L of E to tubes 1 and 2 and 2 L of H to the tubes 3 and 4. (Do NOT add
enzyme to the control tubes A and B.) After adding enzyme, mix by pipetting up and down
about 8 times with a P200 set at about 25-30 L, or finger vortex. (DO NOT MACHINE
VORTEX; enzymes tend to denature under this stress.)
8. Place your tubes in the microfuge and hold down the middle button for a couple of seconds to
collect all of the liquid at the bottom.
9. Make sure that your tubes are closed tightly and place them into the 37C water bath. Avoid
pushing the tubes too far into the rack so that you minimize the risk of bath water leaking into
your tubes.
10. Incubate for at least 25 minutes. During the incubation, proceed to Section B. You might also
use this time to read ahead to later sections, prelabel tubes, and ask us questions if you had
trouble with the prelab exercise.
B. Preparation of the agarose gel
**WEAR GLOVES: THE AGAROSE GEL SOLUTION AND ELECTROPHORESIS
BUFFER CONTAIN ETHIDIUM BROMIDE, A MUTAGEN.**
1. Prepare the gel tray for holding molten agarose by inserting it into the electrophoresis box so
that the red rubber gaskets seal snugly against the sidewalls of the box on the raised platform in
the middle of the container.
2. You should see a purple plastic gel comb next to your electrophoresis box. It has a row of 12
teeth on each side, and the teeth on one side are narrower than those on the other (1 mm vs. 1.5
mm). With the narrower, 1 mm side down, insert the comb into the pair of grooves that are close
to one end of the gel tray. Using the narrower teeth will allow the DNA bands to be relatively
sharp and bright on your gel, making them easier to assess. Before proceeding, make sure that the
comb, tray, and gel box are level so that the poured gel will have a uniform thickness.
3. 60 mL of 0.8% molten agarose will be available in a 50 oC water bath. Carefully pour the
molten agarose into the gel tray.
4. Carefully cover the gel with the electrophoresis tank cover to contain evaporation. Do not
move or jar the comb or casting tray while the agarose solidifies. As the agarose polymerizes (in
10 - 15 minutes), it will change from clear to opaque.
5. After the agarose has hardened, remove the tray from the box, give it a quarter rotation so that
the wells will be closest to the anode (black leads), and replace the tray into the gel box.
6. Gently pour TBE buffer into the gel box until it fills both reservoirs and just barely covers the
entire surface of the gel (no more than 2-3 mm above it).
7. Slowly remove the comb, pulling it straight upwards in order to avoid ripping the wells, and
place it on the large plastic tray provided at your station.
D. M. Dean and J. A. Wilder
10
C. Electrophoresis
You will now run a gel with a portion of each digest to make sure that the digests
worked and to determine the orientations of pUC19 and the pACYC177 Kan fragment
relative to each other in your clone of pUC-Kan.
1. After the restriction enzyme incubation is complete (see Section A), remove the reaction tubes
from the 37oC water bath and place them on ice to ensure that the sticky ends remain denatured.
The size standard, MWS, which will also be loaded into the gel, does not require heating or ice
because it does not have sticky ends.
Note the green coloring within the enzyme reaction buffer and the MWS tube. When you ran
protein gels in 101 and DNA gels in 102, you added a “gel loading buffer”. Such a buffer contains
2-3 different dyes that provide visual markers for how your gel run is progressing. Due to added
glycerol, gel loading buffers are of higher density than water, which ensures that your samples will
not float out of the wells after loading. In these labs, we are using an enzyme reaction buffer that
also contains the components necessary for it to serve as a gel loading buffer, This measure will save
time and tubes.
2. Confirm that the gel wells are on the side of the black electrode. If they are not, rotate the gel
tray 180º and replace it into the tank, adding more buffer to cover it if necessary.
3. With a P20, load the gel lanes as follows (10 l/lane):
Table 4-1. Gel lanes for examining EcoRI and HindIII digests.
Lane
1
2
3
4
5
6
7
8
9
10
11
12
Lab
group
shared
shared
1
1
1
1
shared
2
2
2
2
blank
Tube
contents
to load
A
B
1
2
3
4
MWS
1
2
3
4
Purpose
Undigested
controls
EcoRIdigested
pUCKan
EcoRIdigested
pBR322
HindIIIdigested
pUCKan
HindIIIdigested
pBR322
Size
standard
EcoRIdigested
pUCKan
EcoRIdigested
pBR322
HindIIIdigested
pUCKan
HindIIIdigested
pBR322
PRACTICAL HINTS FOR LOADING GEL:

Steady the micropipette over the well using two hands and with your elbows on the
bench.

Be careful not to punch the tip of the micropipette into or through the bottom of the gel.

Gently depress the micropipette plunger to expel the sample SLOWLY into the
appropriate well. If the tip is centered over the well, the reaction solution will sink to the
bottom of the well. Avoid leakage into neighboring wells.
D. M. Dean and J. A. Wilder
11

Figure 4-2. Loading samples into wells of an agarose gel.
4. Close the top of the gel tank. Do not move or jar the tank, as vibrations may force samples
out of their wells so that they cross-contaminate samples in adjacent wells. As you put the cover
on, connect the red jack on the cover to the red electrode and the black jack to the black electrode.
5. Turn the power supply on, and set it to approximately 100 volts.
6. If you switch the power source to indicate amps, it should now register a significant current
(>a few milliamps). You should also see bubbles rising from both electrodes within the tank.
If no current registers, turn power supply off, check the electrode connections, and try again.
7. Shortly after the current is applied, you should see the dye front (a blue band) moving through
the gel toward the positive (red) electrode. If the dye band moves in the opposite direction,
immediately turn off the power supply, reverse the electrical lead connections on the power
supply, and then turn the power supply back on.
8. Electrophorese for approximately –1-1.5 hours or until the faster-moving (yellow) dye is at the
end of the gel.
9. While the gel is running, proceed to Sections D and E below.
10. Once the gel is done with its run, turn off the power supply and disconnect the electrical
leads.
11. Ethidium bromide has been added to the gel and buffer, so the DNA is labeled and ready to
visualize under UV illumination. Wearing gloves, carefully remove the gel tray and gel from the
box and place into a large plastic weighing boat. Bring the gel to your instructors, and they will
take a photo that you will need for a figure in your lab report. This photo will allow you to
determine the orientation of the Kan fragment relative to the pUC19 vector if you use your
Prelaboratory Exercise 4-1 as a reference.
D. M. Dean and J. A. Wilder
12
Figure 4-3. The sizes in bp for each band of the molecular weight standard (MWS). (Source: Invitrogen).
D. Purifying DNA and Ligation
Once the EcoRI digestion is complete, you will be ligating pUC-Kan and pBR322
together, but first, you will need to purify the DNA from its enzyme buffer and
enzymes. To this end, you will use a purification column made of a modified glass
powder, similar to the one you used in 102 to purify tadpole DNA. DNA associates
with the column, while proteins and other components wash through. After rinsing,
DNA is removed from the column with a solution of low ionic strength, such as
water.
To save tubes, the class will share the same supplies of DNA binding buffer and
Wash buffer. You will find these reagents on the back lab bench. Replace the shared
reagents after use, and always use clean Pipetman tips for each solution.
1. Add 200 L of DNA binding buffer to a clean microfuge tube.
2. Label a DNA purification column (the capped object with a white filter inside) and its
collection tube (the open tube that it sits in) with your initials.
3. After the EcoRI digests are complete and your gel is running, add all 32 L of both Tubes 1 and
2 to the 200 L of DNA binding buffer. Vortex to mix. (A machine vortex is ok because you are
dealing with a large volume and not trying to preserve enzyme function).
4. Pipet all of the DNA and binding buffer onto the filter within the column. Be careful not to
touch the white filter itself as you pipet, since it is fragile.
5. Making sure the column and collection tube are counterbalanced with another purification
column, centrifuge at maximum speed for 60 seconds.
6. Remove your sample from the microfuge, lift the column off of the collection tube, remove the
flow-through with a Pipetman, discard the flow-through, and replace the column into the
collection tube.
D. M. Dean and J. A. Wilder
13
7. Rinse the filter by adding 200 L of Wash buffer onto the column. Spin for 60 seconds in the
microfuge and discard the rinse as you did during Step 6. Replace the column into the collection
tube.
8. Repeat Step 7 once more (i.e. add the 200 L of Wash buffer, spin, discard rinse, replace
column).
9. The Wash buffer contains some ethanol, which can reduce your final yield if not removed.
Without adding any additional liquid to your column, centrifuge for an additional 1 minute to
dry off the excess ethanol.
10. Label a clean microfuge tube “Ligation” along with your initials. Place your column into the
Ligation tube and discard the collection tube.. Add 10 L of ddH2O to the column, taking care to
add the water directly to the column filter without puncturing it. Let the column stand for 1
minute to allow the DNA to be removed. (Don’t worry about closing the tube, since it can’t be
done effectively with the column on top of it.) Spin for 60 seconds.
11. Confirm that the water, which contains your DNA, flowed through the column and into the
Ligation tube, then discard the column if the elution was successful.
12. Add 10 L ddH2O from your Eppendorf tube rack and 2 L LigB from the shared ice bucket
to your Ligation tube. Go to the freezer and add 1 L of T4 DNA ligase, then immediately
replace the ligase into the freezer, since it denatures quickly. Mix your Ligation gently by
pipetting up and down or finger vortexing.
13. Incubate your ligation reaction for 10 minutes at room temperature, then store on ice until it is
needed for transformation.
E. Transformation and plating your Ligation and Control
Earlier during class, you will have been assigned to set up one of the transformation
controls (C-G) that are described below. Make sure that you are clear on which
control you are responsible for.
Unlike the Bacillus cells that we worked with in 101, E. coli cells do not normally
take up DNA from outside of the cell at high efficiency. However, for reasons that
are not well-understood, E. coli that have been treated with calcium chloride and
kept cold are “competent” at receiving foreign DNA. Heat shocking E. coli in this
state induces DNA uptake from outside the cell.
1. Label a clean microfuge tube “Control [your assigned letter] DNA”, prepare your control
plasmids for transformation by mixing the following components:
Table 4-2. Dilution procedures for the transformation controls.
Control
Dilution procedure
C
D
E
F
5 L pUC-Kan1 + 4 L ddH2O + 1 L LigB
5 L pUC-Kan2 + 4 L ddH2O + 1 L LigB
5 L pBR322 + 4 L ddH2O + 1 L LigB
5 L KanRTetRLacZ+ + 4 L ddH2O + 1 L LigB
D. M. Dean and J. A. Wilder
G
14
5 L KanRTetRLacZ-+ 4 L ddH2O + 1 L LigB
2. In your ice bucket, locate two tubes that each contain 100 l of CC (i.e. your competent E. coli
cells). Initial one of these tubes and label it “Lig”. Add 10 l of your Ligation from Section D to
your Lig tube of competent cells. Mix by gently pipetting up and down approximately 6 times
with a P200 set at about 100 l, then place the tube back on ice immediately thereafter to keep the
components cold. Competent cells are very fragile, so do not machine vortex.
3. Initial the other CC tube and label it “Con [letter of your assigned control]”. Add all 10 l of
your diluted Control DNA from Step 1 above to your Control tube of competent cells. . Mix as
you did in Step 2.
4. Incubate your Lig and Con tubes of competent cells and DNA for at least 15 minutes on ice.
5. Bring your ice bucket over to the 37oC water bath, transfer your Lig and Con tubes directly
from the ice bucket into the bath, and incubate at 37 oC for 3 minutes. This “heat shock” will
induce uptake of the plasmid DNA. Avoid pushing the tubes too far into the rack so that you
minimize the risk of bath water leaking into your tubes. While you wait, locate your tube of LB
and be prepared to continue to the next step.
6. Remove the Lig and Con tubes from the water bath and, working quickly, pipet 0.5 ml of LB
into both tubes. Mix by gently pipetting up and down approximately 6 times. Be careful not to
push the P1000 down too far, since the tip is large enough to cause overflow.
7. Cap the Lig and Con tubes tightly and incubate on the 37oC water bath for 30 minutes. Again,
avoid pushing the tubes too far into the rack so that you minimize the risk of bath water leaking
into your tubes. (Discuss: what is this incubation for? Why not immediately plate after heat shocking
cells?)
8. You should have two Kan/Tet/X-Gal (KTX) plates at your bench. Label one of these plates
“Ligation” along with your initials, the date, and which pUC-Kan you started with. Label the
other plate “Control [letter of your assigned control]” along with your initials and the date.
9. After the 30 minute incubation, spin down the cells for 15 seconds in the microcentrifuge at
maximum speed. Remove and discard 450 l of the supernatant into the biohazard bag at your
station.
10. Resuspend the remaining 150 l of cells in the supernatant by pipetting up and down, making
sure the pellet is thoroughly dispersed, then pipet the entire cell suspension onto the appropriate
KTX plate.
11. Rapidly distribute the bacteria over the plates using a sterile spreading rod and turntable
(refer to Lab III and ask us if you've forgotten any details about handling bacteria). Allow the
plates to absorb the cells, agar side down, for 5 minutes at least, then invert the plates and seal
them with Parafilm.
12. Incubate plate (inverted!) for 2 days in the 37 oC incubator.
D. M. Dean and J. A. Wilder
15
13. Before leaving, please throw away any tubes you labeled and processed DNA or cells in, but
keep tubes and reagents that only we labeled for reuse (e.g. toss the CC tubes but not E). Wipe off
the countertop with ethanol and wash hands. Finally, watch a demo for what you will need to do
on Day 3 (see Section F below).
Day 3
F. Inoculate liquid media with a transformed colony
**DO THIS PROCEDURE BEFORE 4PM, 2 DAYS AFTER YOUR LAB DAY.**
Remember to use sterile technique throughout. Only one culture tube is necessary per
pair.
1. Examine both your Lig and Con plates and write down basic observations. Did you get what
you expected in both cases? Contact us with any questions.
2. In the fridge to the right of the incubators, obtain a liquid sample of LB/Kan/Tet (5 ml) in a
culture tube. Label this tube with your name and date using the masking tape provided.
3. Select a well-separated colony on your Lig plate. Write down whether this colony is blue or
white. Using a sterilized metal loop, pick this colony, open the culture tube, and inoculate the
media with the colony. If you do not have transformants, obtain a colony from a colleague or
from our backup plates on the bench across from the small fridge. SOME BACKUPS ARE
DERIVED FROM pUC-KAN 1 AND SOME FROM pUC-KAN 2. MAKE SURE THAT YOU
CHOOSE THE RIGHT BACKUP SO THAT YOU CAN STILL USE YOUR WEEK 1 DATA,
AND AGAIN, WRITE DOWN IF THE COLONY YOU USED WAS BLUE OR WHITE.
4. Reseal your Lig plate with Parafilm and store both your Lig and Con plates inverted in the
fridge, within a bin that has been labeled with your lab day.
5. Place the inoculated tube back into the fridge in a rack labeled for your lab day.
After 4PM, we will incubate this rack of tubes in a 37°C shaking water bath overnight and
replace it in the fridge the next morning. We will isolate recombinant plasmids from these
cultures next week in lab.
D. M. Dean and J. A. Wilder
16
Week 2
Today, you will isolate your recombinant plasmid from the Day 3 liquid culture and
subject this plasmid to restriction mapping and PCR. Work with the same partner as
last week.
A. Isolation of the recombinant plasmid
This is a very similar procedure to the glass powder-based kit you used during Part 1, but this kit
is designed to lyse cells and extract plasmids from other cellular components, while the other kit
purified DNA from a relatively simple solution of enzymes and buffer. To save tubes, the class will
share the same supplies from the kit . You will find these reagents on the back lab bench. Replace
the shared reagents after use, and always use clean Pipetman tips for each solution.
All centrifuge steps are to be done with the instrument set at maximum speed.
1. Find your liquid culture and vortex to resuspend the cells. Make sure that the cells are
thoroughly resuspended before proceeding.
2. Label a clean microfuge tube with your initials, then add 1.5 mL of liquid culture to your tube.
Making sure the tube is counterbalanced, centrifuge for 60 seconds. Remove and discard the
supernatant by pipetting.
3. Add 1.5 mL more of culture, spin, and discard supernatant twice more in order to pellet most
or all of the 5 mL culture into the same tube. Make sure that as much supernatant as possible is
removed before proceeding, even if a small portion of the pellet is drawn up in the process.
4. Add 250 L of P1 buffer to the cells, then resuspend the pellet by scraping it with a pipet tip,
pipetting up and down, and machine vortexing. Make sure that the cells are thoroughly
resuspended before proceeding, because pelleted cells will not lyse efficiently. (In this procedure,
RNA can be copurified with plasmids, and this would interfere with our experiment. To get around this
problem, P1 is a DNA-friendly buffer that contains RNAse.)
5. Add 250 L of P2 buffer. Invert 8 times to mix, but DO NOT VORTEX at this step. (P2 is an
alkaline, detergent solution that will lyse the cells, and vortexing would shear chromosomal DNA into
smaller fragments, causing it to be co-isolated with the plasmid.) Proceed immediately to the next step.
6. Within a minute after performing Step 5, add 350 L of N3 buffer and mix immediately by
inverting 8 times. (Do NOT vortex.) It is necessary to add N3 quickly, because the plasmid may
degrade if left in alkaline solution for more than a few minutes. (N3 neutralizes the alkaline
solution.)
7. Centrifuge for 10 minutes at maximum speed.
8. Transfer the supernatant to a clean microfuge tube that is labeled with your initials. Avoid
disturbing the pellet, but it is ok if some insoluble debris is transferred as well. Centrifuge the
transferred supernatant for another 5 minutes.
9. Initial your spin column and collection tube. Apply the supernatant from Step 8 to the spin
column.
D. M. Dean and J. A. Wilder
17
10. Centrifuge for 60 seconds. Remove the column, withdraw the flow-through with a P1000,
discard the flow-through down the sink, and replace the column.
11. Add 0.5 mL PB buffer to the column and spin 60 seconds to rinse the column of non-DNA
material. As you did in Step 10, remove the column, discard the flow-through, and replace the
column.
12. Add 0.75 mL PE buffer and spin 60 seconds to rinse the column further. Remove the column,
discard the flow-through, and replace the column.
13. The PE buffer contains some ethanol, which can reduce your final yield if it is not removed
from the filter before elution. Without adding any additional liquid to your column, centrifuge
for an additional minute to dry off the excess ethanol.
14. Initial a fresh microfuge tube and label it “Final plasmid”. Also write on the tube whether you
started with pUC-Kan1 or 2. This will allow us to use this tube as a backup in future years. Discard the
collection tube and place the purification column into a fresh microfuge tube. Add 50 L of sterile
ddH20 directly to the column, taking care not to damage the filter with your pipet tip. Let the
column stand for 1 minute to allow the DNA to be eluted.
15. Centrifuge for 1 minute to elute the DNA. After making sure that you have flow-through in
your microfuge tube, discard the column and close the tube.
B. Restriction mapping
1. In your ice bucket, find a tube of 10X concentrated enzyme reaction buffer (“EB”), HindIII
enzyme (“H”), and BamHI enzyme (“B”). Label three clean microfuge tubes “H dig”, “B dig” and
“Control” and initial them all.
2. Add 4 L ddH2O, 2 L EB, and 14 L of your isolated Final plasmid to each of these three
tubes. Add 1 L of H to your H dig tube and 1 L of B to your B dig tube. Do not add any
enzyme to your Control. Mix gently by pipetting up and down or finger vortexing.
3. Make sure that your Final plasmid tube is clearly labeled and replace it into your ice bucket.
4. Place the three tubes into the 37C water bath for at least 25 minutes. As the digest progresses,
proceed with Section C below.
5. After the incubation is complete, heat your samples at 70C for 10 minutes to inactivate the
enzyme.
6. Place your samples into the microfuge and hold down the middle button for a couple of
seconds to collect all of the liquid at the bottom, then place the three tubes within your ice bucket.
We will collect them after class and freeze them. Next week, you will load these samples into an
agarose gel.
C. Polymerase chain reaction
We will use two PCRs to analyze our recombinant plasmids. One reaction will use the primers pUC1 and
Tet (“Mix 1”), and the other will use pUC2 and Tet (“Mix 2”). Each group of four will be assigned one of
the two reaction mixes to make, but then we will share so that every group uses both Mixes.
D. M. Dean and J. A. Wilder
18
1. Using a clean microfuge tube, make a 1:1000 dilution of your Final plasmid (i.e. the
remaining, undigested portion) in ddH2O. Check the dilution scheme with us if you are at all
unsure as to how to set this up.
2. Place another clean microfuge tube on ice and label it Mix 1 or Mix 2, depending on which mix
you were assigned. Within a shared ice bucket on your bench, you should find all of the PCR
components described in Table 4-3 (except ddH2O, which is within your Eppendorf tube rack).
Add the following components, keeping the tube on ice as much as possible. Mix after adding
each component by pipetting up and down with a P200 and/or finger vortexing.
Table 4-3. PCR mix recipes
Component
Volume (L)
ddH2O
5X PCR buffer
25 mM MgCl2
10 mM dNTP mix
Taq DNA polymerase (5U/L)
16 M Tet primer
49.5
20
10
2
1
6.25
for Mix 1 only: 6.25 pUC1,
for Mix 2 only: 6.25 pUC2
16 M pUC primer
3. After thoroughly mixing the components. Place your Mix into the microfuge and hold down
the middle button for a couple of seconds to collect all of the liquid at the bottom, then
immediately replace it into the ice bucket.
4. Label two small PCR tubes with your initials, and then label one of these tubes 1 and the other
2. To avoid rubbing the lettering off, try and fit all of this information on the side, between the
two ridges near the top of each tube. Add 19 L of Mix 1 to PCR tube 1. Add 19 L of Mix 2 to
PCR tube 2.
5. Add 1 L of your diluted (1:1000) Final Plasmid to each PCR tube. After adding the DNA, set
the P20 at 15-20 L and mix by pipetting up and down. Gently tap the PCR tubes on the
countertop to collect the liquid at their bottoms.
6. Leave your PCR 1 and 2 tubes on your ice bucket with your restriction digests (B dig and H
dig) and the remainder of your undigested Final plasmid. We will set the PCR going overnight
but will show you a quick demo of the PCR machine before you leave. Discard the other tubes
that you have labeled, but leave the tubes that we have labeled (H, B, EB, etc.).
D. M. Dean and J. A. Wilder
19
Prelaboratory Exercise 4-3
It remains to run a gel in order to visualize your restriction map and PCR from Part 2.
However, before lab, it is necessary to know where your PCR primers anneal so that you
can deduce the possible outcomes.
Turn in this exercise at the start of the Week 3 lab.
Recall our use of Pubmed two weeks ago. The National Center for Biotechnology Information
(NCBI) gave us this resource, and they have many other tools available on their website. We will
use other NCBI tools to determine where our PCR primers anneal.
You will be emailed the primer sequences to facilitate copying and pasting them into BLAST, but
for your reference, here are the primer sequences in hard copy:
pUC1 5’-TCACTCATTAGGCACCCCAGGC-3’ (anneals to pUC19)
pUC2 5’-ATCAGGCGCCATTCGCCATTC-3’ (also anneals to pUC19)
Tet
5’-CGCCATAGTGACTGGCGATGCTG-3’ (anneals to pBR322)
1. Log onto the NCBI homepage (http://www.ncbi.nlm.nih.gov/). Click on “BLAST” at the top
right.
BLAST is a toolkit, giving you ways to compare DNA and protein sequences to each other.
Essentially, you can take a sequence of interest and ask what it is similar to. Take a look at the
options on your current webpage. Under Basic BLAST, you should see several options for a oneway query. You can take a nucleotide sequence and ask for similar published nucleotide sequences
(Nucleotide BLAST). You can do the same with a protein sequence, using it to look for similar
protein sequences (Protein BLAST). Alternatively, you can mix and match, taking, for example,
a protein sequence and asking if a DNA sequence elsewhere (say, in another species) might encode
a similar protein (BLASTx). However, we will do a direct, pairwise comparison of two sequences
called BLAST2. You will ask the server where our PCR primers align on our vectors so that you’ll
know what the expected PCR product sizes may be when you examine your actual results.
2. At the bottom of the page, under Specialized BLAST, click on “Align two or more sequences
using BLAST”. You should now see two windows asking you for Sequence 1 and Sequence 2.
3. Copy and paste the pUC1 primer sequence into the Sequence 1 window.
4. Type the pUC19 accession number (L09137) into the Sequence 2 window and click on BLAST
at the bottom left.
5. On the line below, record the positions of pUC19 that the primer anneals to as well as the
relative orientation of the primer to the template (example shown below). Note that BLAST2 will
show you multiple alignments, including random matches of 6-10 bp of sequence. Only record the
full-length alignment between the 20-22 bp primer and the template (i.e. the alignment that is
presented closest to the top of the page).
_________________________________
(over)
D. M. Dean and J. A. Wilder
20
Figure 4-3. Example of BLAST2 alignment between a primer (“Query” on top row) and a
template (“Sbjct” on bottom row). The 5’ end of the primer corresponds to nucleotide #251 of the
published template sequence, and here, the primer and published template sequences run in the
same direction. However, note that the primer may run in the opposite direction in other cases.
6. Repeat this procedure for the pUC2 primer and pUC19 template.
_________________________________
7. Recall that the plasmid containing the Tet gene was pBR322 (accession number J01749), Paste
the Tet primer sequence into the Sequence 1 window of BLAST2 and J01749 into the Sequence 2
window and BLAST. On the line immediately below, record the positions of pBR322 that the
full-length primer anneals to as well as the relative orientation of the primer to the template.
_________________________________
D. M. Dean and J. A. Wilder
21
Week 3
Today, you will separate your Week 2 restriction fragments and PCR results by size
using agarose gel electrophoresis.
Work with the same partner as the previous two weeks, but a group of four shares a gel.
**WEAR GLOVES: THE AGAROSE GEL SOLUTION AND ELECTROPHORESIS BUFFER
CONTAIN ETHIDIUM BROMIDE, A MUTAGEN.**
1. Prepare an agarose gel for loading as you did on Week 1 of this lab, Section B of the procedure.
2. Locate your restriction digests (B dig and H dig), your digest Control, and your PCR tubes (1
and 2). Every group of four should also have a molecular weight standard (MWS) within an
Eppendorf tube rack. Make sure that the contents of all of your tubes are completely thawed
before proceeding.
3. Place B dig, H dig, and the Control into a 37 oC water bath for about 3 minutes to denature the
sticky ends, then immediately chill the tubes on ice until you are ready to load them into the gel.
The MWS and PCR tubes do not require heating, since they do not have sticky ends. Note that, as in
Week 1, the restriction enzyme and PCR buffers already contain the components necessary to
double as gel loading buffers.
4. Using all 20 l of the Control, B dig, and H dig tubes, and only 10 l of PCR1, PCR2, and
MWS, load the gel lanes in the following order:
Table 4-3. Gel lanes for restriction map and PCR results
Lane
1
2
Lab
Group
Tube
3
4
5
1
Control
B dig
H dig
6
7
8
both
PCR 1
PCR 2
MWS
9
10
11
2
Control
B dig
H dig
12
blank
PCR 1
PCR 2
5. Turn the power supply on, and set it to about 100 volts. Run the gel as you did on Week 1 of
this lab, Section C of the procedure.
6. While the gel runs, take a moment to re-examine the results from your experimental KTX plate
and all of the plated controls that we did as a class. Discuss with us if any of the results don’t
make sense. This is also a good time to discuss strategies for deriving and presenting your Final
Plasmid maps.
7. Once the faster-moving (yellow) dye is at the end of the gel, turn off the power supply and
disconnect the electrical leads.
8. Wearing gloves, carefully remove the gel tray and gel from the box and place into a large
plastic weighing boat. Bring the gel to your instructors, and they will take a photo that you will
need for another figure in your lab report. This photo will allow you to determine the relative
orientation of the DNA fragments within your Final Plasmid.
Directions for the lab report are on the next page.
D. M. Dean and J. A. Wilder
22
Lab report writeup
This assignment will be formatted as a formal lab report. See the Appendix for details on this
format. In your report, please answer the following questions:
1) Materials and methods section: in no more than 1.5 double spaced pages, summarize the major
steps in the experimental procedure. To keep it brief, don’t get into too many technical details,
such as talking about temperature, times, and spinning speeds. We want to see here that you
understand why each major step is important, not so much that you can recall each detail of a step. If you
had to do any particular step differently than instructed, mention this here.
2) Draw maps for pUC19, pACYC177, the two possible pUC-Kan plasmids, and pBR322,
including all of the features that you were asked to find in Prelaboratory Exercise 1. Discuss the
possible results for all of the EcoRI and HindIII digests from Week 1, describe your particular
agarose gel results, and then tell us which pUC-Kan plasmid you think you have. Include the
picture of your Week 1 gel with the lanes labeled and a descriptive figure legend.
3) Draw maps of and describe the two possible plasmids that you would get if one pUC-Kan and
one pBR322 molecule were to ligate together and form a circular plasmid. (Check in with us if
you think you have something different from this type of vector.) Include all of the features that
you were asked to find in Prelaboratory Exercises 1 and 3. For each of these two plasmids,
discuss the possible digest and PCR results from Week 3, describe your particular agarose gel
results, and then tell us which Final Plasmid you think you have. Include the picture of your
Week 3 gel with the lanes labeled and a descriptive legend. Incorporate the KTX plate results
(including the controls) in your discussion.
4) Did any of your reactions fail? Any other odd results or procedural difficulties? Discuss specific steps
you might take to resolve any lingering issues. Did different samples suggest different Final Plasmid
maps? If so, interpret each sample separately, tell us which data you believe more and why, and
then give us your best guess for a plasmid map with the data that you have.
Reference
Maniatis, T., Fritsch, E.F., and J. Sambrook (1987). Molecular Cloning: A Laboratory Manual.
Cold Spring Harbor, NY
Also used standard protocols from Zymo Research, Qiagen, New England Biolabs, and Fermentas.