Your introduction should probably be around 1 – 1.5 pages in length. Basically, here you summarize IN YOUR OWN WORDS the material in the introduction to the exercise. This is the best place to get in your references to primary literature too. You may want to briefly talk about the history of cloning. I highly recommend looking up terms, such as cloning methods and uses, on Wikipedia, and then using the references in that article (you must actually go to the referenced literature and read it though).
I attached the lab manual it will be on exersice 3 and 4
lab_mann_exercise_3_4.docx
bio366l_manual_v49.pdf
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Exercise 3 Basic Cloning Methods
Introduction
The study of genes and their products has been revolutionized by the ability to isolate any particular
gene from its environment, a chromosome, so that it can be analyzed by itself. These days, cloning is a
routine laboratory method. Three basic advances contributed to cloning as we know it today: the
discovery of restriction enzymes, which allow the fragmentation of DNA at specific sites; the ability to
propagate individual genes on small DNA molecules, known as plasmids, which replicate independently;
and the ability to selectively increase the amount of DNA coding a desired gene or gene fragment with
the polymerase chain reaction. Each of these three elements will be discussed in turn. The rationale
behind cloning is to cut a chromosome containing an interesting gene into small pieces, one of which
will contain the gene and inserting this gene into a plasmid shuttle, or vector, which replicates
independently of the original chromosome. Restriction enzymes are proteins that cut DNA. There are
several general types of restriction enzymes: the Type II restriction enzymes are the most commonly
used in cloning. Each of these type II restriction enzymes recognizes a particular sequence, generally
ranging in size from 4 to 8 base pairs long, binds to this DNA recognition sequence, and cuts each DNA
strand at a specific place within the sequence. Some of these enzymes leave what are called blunt ends,
because both DNA strands are equally long. Such enzymes are not quite as convenient to work with as
enzymes that leave “overhangs” or “overhanging ends.” Overhangs describe DNA ends in which one
strand is longer than the other, and thus overhangs the shorter end. Any given enzyme will leave the
same ends every time it cuts (at least 99% or higher; there are always exceptions to everything in
biology). Such ends are complementary to each other: any pair of ends created by a single restriction
enzyme can re-anneal to each other (see below).
5’—xxxxGGATCCxxxx—3’ xxxxGGA TCCxxxxx Blunt ends 3’—xxxxCCTAGGxxxx—5’ xxxxCCT AGGxxxxx
5’—yyyyAAGCTTyyyy—3’ yyyyA AGCTTyyyyy Sticky ends 3’—yyyyTTCGAAyyyy—5’ yyyyTTCGA Ayyyyy
Cutting a chromosome with a given restriction enzyme is only the first step of the cloning process. This
first step allows the isolation of an interesting piece of DNA from a whole bunch of uninteresting (at
least at a given moment) DNA. The interesting piece of DNA now must be inserted into a smaller piece
of DNA, known as a vector, which replicates independently of the chromosome. There are several types
of vectors, each with their uses, pros and cons, but they all share some important characteristics: they
have their own origin of replication; they carry an antibiotic resistance gene, or another gene that acts
as a “marker” for the vector – this marker lets us track the vector, like a Federal Express tracking
number; and finally they have a place into which new DNA can be inserted, such as some convenient
restriction enzyme sites, present only once in the vector DNA. Modern vectors now contain a
“polylinker,” which is simply a small stretch of DNA which contains as many as 10 – 15 sites for different
restriction enzymes, giving us a choice of enzymes to use in the cloning process. High copy number
vectors are useful because they yield
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high amounts of DNA; when one needs to purify a protein, for example, that can be very useful. On the
other hand, the inserted gene may be toxic to the host cells; in this case, a low copy number vector is
essential. Vectors that carry large inserts (up to 200 kb) are useful in genome mapping and sequencing
projects, so that large sections of chromosomes can be analyzed as a chunk. Most vectors are
propagated in bacteria (predominantly E. coli), since they can be grown cheaply and to very high
numbers. Yeast has also been successfully used as host cells.
Table 1. Commonly used vectors
Vector name Vector type Size of insert Copy number pBR322 plasmid 100 bp – 10 kb medium (20)
pUC19, pKS, etc. plasmid 100 bp – 10 kb high (500) lambda phage 3 – 10 kb regulatable (1 – 100 )
cosmid phage/plasmid up to 40 kb high (20 – 100, dep, on size) PAC phage artif. chrom. up to 100 kb
regulatable (1 – 100 ) BAC plasmid artif. chrom. up to 200 kb low YAC yeast artif. chrom. 100 – 500 kb 1
–2
To make the final vector, the two pieces of DNA have to be linked together. The enzyme ligase, which
normally ligates Okazaki fragments together during DNA replication, is used for this purpose. Ligase, an
ATP-dependent enzyme, is quite inefficient in aligning two DNA ends together. This is where
overhanging, or sticky, ends are a great advantage: because the overhangs are complementary, they
anneal and “stay” together long enough for ligase to seal the nicks in DNA. Blunt end ligation relies on
the rare occurrence when the DNA molecules happen to align appropriately. Ligase also requires a 5′
phosphate at the end of one DNA strand and a 3′ OH group at the end of the other strand: it then
carries out a condensation reaction in which water is released after ligation is accomplished.
What happens if the piece of DNA you want to clone is very rare, or if it does not have useful restriction
sites for cloning? This is where PCR has turned out to be a wonderful tool. PCR allows the selective
amplification (replication) of one segment of DNA (Fig. 1). During amplification, you can attach
“handles” to the DNA segment even if they were not present originally. The catch is that you need to
know something about the piece of DNA you are amplifying, namely two small stretches of DNA (15-20
bp) on either side of the DNA you are trying to clone. Using these known sequences, you will design two
primers, which will be extended during many rounds of DNA replication, eventually leading to many
copies of the DNA segment between the two primers. The primers must be identical to the template
DNA (the chromosome which acts as the master copy) at their 3′ end, but if the primer is
complementary to the template for 15-18 base pairs, some DNA not present in the original sequence
can be added to the 5′ end of each primer. These “handles” can be designed to contain the desired
restriction enzyme site at each end. After PCR, the resulting PCR products are cut with this restriction
enzyme and then ligated to vectors that have complementary overhanging ends.
One last problem before the experiment is done: how do you know when you have the right clone? As
mentioned above, ligation is quite inefficient, so only a small proportion of vector
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molecules will have inserts, not to mention the right insert. After all, the point of the whole experiment
is to isolate the piece of DNA you want from all the other stuff. It is nearly impossible to get enough
ligated DNA to analyze this DNA directly after ligation. We therefore use the power of host cells, which
will replicate the DNA to high numbers (think about a billion Escherichia coli [E. coli] cells per mL, each
with 200 copies of a plasmid). So the first thing to do after a ligation is to transform (introduce) your
ligation mixture into a host strain, generally E. coli cells. E. coli has a very useful property: its nucleases
destroy linear DNA; only circular DNA survives for any reasonable length of time. So in fact, only vectors
that have been ligated with an insert and vectors that were never cut can survive and become
established in E. coli cells. How do you tell one from the other? If you are lucky, the insert you are
cloning may have its own phenotype, which can be selected directly (for example, you are cloning an
antibiotic resistance gene not present on the vector DNA). Since the insert cannot replicate on its own
without the origin of replication from the plasmid, you will automatically get vectors carrying inserts.
Otherwise, the only way you can tell is by analyzing the size of the vector – if it has increased by the size
of the piece of DNA you added then you’re home free. One alternative to this is to use a screen – a test
which distinguishes between cells which carry vectors with inserts and cells which carry vectors without
inserts (for example the “blue-white” screen). In this exercise the gene to be cloned is a gene that
confers E. coli with antibiotic resistance to the antibiotic, gentamicin. Some antibiotics are
bacteriostatic while others are bactericidal. Bacteriostatic antibiotics stop bacteria from reproducing.
On the other hand, bacteriocidal antibiotics actually kill bacteria. Gentamicin is a bacteriocidal
antibiotic. If, after transferring the gene for antibiotic resistance, cells are immediately spread on an
agar plate with the antibiotic the transformed cells may not have time to sufficiently express the gene
that confers the antibiotic resistance. This would result in the death of the transformed cells and no
copies of the gene. To bypass this potential problem the cells are typically put into an “ultra” rich
medium such as SOC without an antibiotic and incubated at optimal growth temperature for 30 – 90
minutes immediately after introducing the gene into E. coli.
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Methods and Analysis
This exercise and the remaining exercises involve a great deal of sterile work. Please be sure to cover
sterile pipette tips and eppendorf tubes immediately after use so that others will also have sterile
supplies.
A. Preparation of PCR products (use sterile pipette tips and be as accurate as you can) Each group
should set up one PCR reaction tube.
1. Label the top of a sterile 0.2 mL tube with a group identifier; put the tube on ice and add 47 μL of the
PCR rxn solution to each tube. 47 μL of this solution is made up of the following:
35.62 μL sterile Nanopure water 5.0 μL 10X PCR reaction buffer 1.0 μL dNTPs (10 mM each) 2.5 μL
forward primer (20 μM stock) 2.5 μL reverse primer (20 μM stock) 0.38 μL AmpliTaq enzyme2
2. Add 3.0 μL of the plasmid template (500 pg/μL) to the PCR rxn solution and gently pipet the mixture
up and down to mix.
3 Make sure to keep the reaction mixture on ice until you are ready to load it into the thermocycler (PCR
machine).
4. Program the thermocycler for the following cycles:
a. An initial period of 5 minutes at 95°C (hot start activation) b. 30 cycles of – 45 seconds at 95°C
(denaturation) – 45 seconds at 58°C (annealing) – 1 minute at 72°C (extension/elongation) c. A final
elongation step of 5 minutes at 72°C (to finish any unfinished products) d. 4°C holding step
For day 2 of Exercise 7, you will split your group into two. Half of the group will perform a PCR
purification and a digestion to prepare the insert for cloning (continue with step A5 to A6). The other
half of your group will perform a miniprep to purify the vector and a digestion to prepare the vector for
cloning (start with Part B).
5. After the PCR reactions are done, you need to perform a quick purification using the Invitrogen
PureLink PCR Purification Kit (Appendix 4); this step gets rid of excess primers and the buffers of the PCR
reaction, which may inhibit the restriction enzyme digest.
2 The AmpliTaq enzyme was kindly donated by Thermo Fisher Scientific, Inc.
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Transfer the PCR products from the 0.2 mL PCR tube to a single 1.5 mL microcentrifuge tube and then
follow the directions in Appendix 4.
6. Restriction digest of the PCR products
a. Assemble the following reaction in a 1.5 mL microcentrifuge tube:
____ 9.4 μL Purified PCR products (using method from Appendix 4) ____ 1.1 μL 10X restriction
enzyme buffer (10X FastDigest Buffer)3 ____ 0.5 μL HindIII restriction enzyme (FastDigest HindIII)5
Mix well by pipetting up and down several times and by using the tip as a stirrer. Avoid introducing
bubbles while mixing. Restriction enzymes are stored in 50% glycerol, which makes them much denser
than water; when you add them to a tube, they will drop straight to the bottom of the tube. Without
thorough mixing, there will not be efficient digestion because the enzyme will not be distributed evenly
in the reaction tube.
b. Label the tube as “PCR product digest.” Additionally, label it with a group identifier and a section
number. Place the tubes in a 37°C incubator or a 37°C water bath for 20 minutes and then store it in the
refrigerator until the next lab.
B. Preparation of the vector DNA
1. The Invitrogen PureLink® Quick Plasmid Miniprep Kit is used to isolate vector plasmid from bacteria.
Add 1.4 mL of overnight culture containing your plasmid to a 1.5 mL microcentrifuge tube and spin the
cells in a microcentrifuge at top speed (15,000 RPM) for 1 minute (no longer); make sure you use
another tube as a balance.
2. Remove and discard all of the supernatant with a Pipetman fitted with an appropriate pipette tip, but
do not disturb the pellet. Discard the supernatant into the bottle labeled “Luria Broth with E. coli
disposal.” Discard no other solutions into this container during this preparation.
3. Resuspend these cells in 250 μL of Solution R3; resuspend carefully and completely by using a pipette
tip to dig into and disperse the cell pellet; if you do not resuspend the cells well, the following step will
be inefficient and your yield of plasmid DNA will be very low.
4. Add 250 μL of Solution L7; DO NOT VORTEX; mix gently by inverting the tubes 4-5 times – lysis will be
seen by a clearing of the solution (intact cells are opaque, lysed cells are translucent) and an increase in
the viscosity of the solution as the cellular DNA is released (solution will appear snotty). Incubate at
room temperature in Solution L7 for 2-3 minutes (make sure that this incubation does not proceed
longer than 5 minutes).
3 10X FastDigest Buffer and FastDigest HindIII are tradenames for products from Thermo Fisher
Scientific, Inc. These products have been developed such that they result in a faster DNA digestion than
a standard HindIII digestion.
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5. Add 350 μL of Solution N4; MIX BY INVERTING THE TUBE GENTLY; if all has gone well up till now, this
solution will cause a lot of white precipitate to form.
6. Spin tubes in the microcentrifuge at 11,200 RPM (12,000 x g) for 10 minutes. While the cell debris is
pelleting, prepare the spin apparatus by inserting the spin column (clear/white column) into the 2 mL
wash collection tube.
7. Once the cell debris is in the pellet, carefully transfer the clear supernatant to the miniprep spin
column using a P1000 Pipetman. Avoid taking up any of the white precipitate; if you did not get a good
pellet, spin again before doing this step.
8. Spin the column in the microcentrifuge for 1 minute at 11,200 RPM (12,000 x g) and discard the flow
thru in the collection tube.
9. Put the spin column back into the wash collection tube, add 500 μL of Wash Buffer W10 to the spin
column, and spin at 11,200 RPM (12,000 x g) for 1 minute. Discard the flow thru and place the spin
column back into the wash collection tube.
10. Add 700 μL of DNA Wash Buffer W9, spin at 11,200 RPM (12,000 x g) for 60 seconds, and again
discard the flow thru. Place the spin column back into the 2 mL wash collection tube. 11. Spin the
empty column for 1 minute in the microcentrifuge at 11,200 RPM (12,000 x g). This dries the spin
column filter. This is an important step to make sure that no ethanol is carried over into the final
purified plasmid.
12. Remove the spin column from the wash collection tube and place the spin column in a new clean,
sterile 1.5 mL microcentrifuge tube (recovery tube).
13. Add 35 μL of sterile nano-water directly to the top of the spin column filter to elute the DNA.
Incubate the spin column at room temperature for 2 minutes, then spin in the microcentrifuge for 1
minute at 11,200 RPM (12,000 x g). The eluate is your plasmid DNA to be used as vector for cloning.
Note: What does R3, L7, N4, W10, and W9 mean, and what is the purpose of each?
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14. In a 1.5 mL microcentrifuge tube assemble a restriction digest reaction as follows:
8.9 μL vector DNA (just isolated); 1.1 μL 10X restriction enzyme buffer (10X FastDigest Buffer) 0.5
μL HindIII restriction enzyme (FastDigest HindIII) 0.5 μL calf intestinal alkaline phosphatase (FastAP)4
Mix well by pipetting the mix up and down several times.
15. Close the tube tightly and incubate in a 37°C incubator or a 37°C water bath for 20 minutes. After
the incubation, store the tube in the refrigerator until the next lab meeting.
16. While the restriction digest is incubating, add 1.1 μL of 10X restriction enzyme buffer to 9.9 μL of
vector not used in the restriction digest; save this uncut plasmid DNA in the refrigerator at 4°C. Discard
the remainder of the uncut vector.
C. Agarose gel electrophoresis to separate the products of the restriction enzyme digests
1. Position a gel-pouring tray in a gel caster apparatus as shown to you by the instructor. Place it in the
fume hood. Using the small round level adjust the apparatus screws until the tray is level.
2. Make up 20 mL of 0.8 % agarose in TAE (Tris/acetic acid/EDTA) buffer in a 50 mL Erlenmeyer flask.
Microwave the 0.8 % agarose in TAE at a power setting of 6 for 42 seconds. If not all the agarose
crystals dissolved, then microwave for a few more seconds. Carefully watch the flask in the microwave
during heating. Do not let the agarose mixture boil over the top of the flask. After dissolving the
agarose in the microwave oven, allow the solution to cool down until it does not burn when touched.
Pour the solution into the gel-casting tray and position the gel comb in the slots on the top of the gelcasting tray.
3. Allow the solution to harden for at least 45 minutes before carefully removing the gel comb.
4. Immerse the gel with the gel-casting tray into the electrophoresis apparatus; add running buffer (TAE)
until it just covers the gel completely.
**Note: steps 1-4 may be performed by the management.**
5. Take out your sample tubes and briefly (5 seconds at > 1,000 RPM) spin down your sample tubes [1.
Digested PCR insert; 2. Digest vector; 3. Undigested vector].
6. Add 2.2 μL of 6X DNA loading dye to each of the sample tubes.
4 FastAP is a tradename for an alkaline phosphatase from Thermo Fisher Scientific, Inc. Since this
phosphatase uses the same buffer as FastDigest HindIII, the vector DNA may be simultaneously digested
with HindIII and dephosphorylated by the alkaline phosphatase.
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7. The DNA loading dye solution fills two crucial functions: it increases the density of the sample so that
it sinks to the bottom of the well when it is loaded on the gel, and it allows tracking during the
electrophoresis.
8. Carefully load all of the digested vector into one well and all the digested PCR insert into another well
of the gel. Additionally, each gel should have 6 μg of the 1 kb DNA ladder (0.038 μg/μL) and a well
containing the undigest vector. If running a large gel, the DNA ladder may be loaded into multiple wells.
9. Connect the leads such that the negative pole (black lead) is nearest the gel wells and the positive
pole is nearer the opposite end of the gel.
10. If using the 8-lane gel, set the voltage to 120 V. If using the 15-lane gel, set the voltage to 90 volts.
Run the gel for 45 minutes. The dye front should be about 1 centimeter from the bottom of the gel. Do
not let the dye run off the gel.
11. The size (kb) of DNA represented by each of the bands in the Thermo Scientific GeneRuler 1 kb Plus
DNA ladder @ concentration of 0.5 µg/µL (Cat # FERSM1331) is as follows:
12. Slide the gel from the gel tray into an appropriately sized plastic container and pour enough of the
Sybr Green Stain5 over the gel so that the gel is submerged about one centimeter
5 The Sybr Green Stain was generously donated by Thermo Fisher Scientific, Inc.
Photo courtesy of Thermo Fisher Scientific.
Size (bp) ng/0.5 μg %
20,000 20 4 10,000 20 …
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