I need you to write introduction for these three experiment (Combine it together) and write the Material and Method for the three experiment. You don’t need to include all the details in the material and methods. Please follow the diresction like the following:Introduction1- Write introduction for ex 3 and include outside reference 2- Introduction for ex 4 include outside reference3- Write a summary for ex 5Material and method: Write summary of material and methods for ex 3Then write a summary for ex 4 Then write a summary for ex 5Please follow the Rubric and Attachment is the lab manaul that you will copy write from.
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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’
3’—xxxxCCTAGGxxxx—5’
xxxxGGA TCCxxxxx
xxxxCCT AGGxxxxx
Blunt ends
5’—yyyyAAGCTTyyyy—3’
3’—yyyyTTCGAAyyyy—5’
yyyyA AGCTTyyyyy
yyyyTTCGA Ayyyyy
Sticky ends
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
pBR322
pUC19, pKS, etc.
lambda
cosmid
PAC
BAC
YAC
Vector type
plasmid
plasmid
phage
phage/plasmid
phage artif. chrom.
plasmid artif. chrom.
yeast artif. chrom.
Size of insert
100 bp – 10 kb
100 bp – 10 kb
3 – 10 kb
up to 40 kb
up to 100 kb
up to 200 kb
100 – 500 kb
Copy number
medium (20)
high (500)
regulatable (1 – 100 )
high (20 – 100, dep, on size)
regulatable (1 – 100 )
low
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
5.0 μL
1.0 μL
2.5 μL
2.5 μL
0.38 μL
sterile Nanopure water
10X PCR reaction buffer
dNTPs (10 mM each)
forward primer (20 μM stock)
reverse primer (20 μM stock)
AmpliTaq enzyme
2
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.
Page 42
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)
____ 0.5 μL HindIII restriction enzyme (FastDigest HindIII)5
3
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.
Page 43
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 gel-casting 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.
Photo courtesy of Thermo Fisher Scientific.
11. The size (kb) of DNA represented by each of the bands in the Thermo Scientific GeneRuler
1 kb Plus DNA ladder @
Size (bp)
ng/0.5 μg
%
concentration of 0.5 µg/µL
(Cat # FERSM1331) is as follows:
20,000
20
4
10,000
20
4
7,000
20
4
5,000
75
15
4,000
20
4
3,000
20
4
2,000
20
4
1,500
80
16
1,000
25
5
700
25
5
500
75
15
400
25
5
300
25
5
200
25
5
75
25
5
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 …
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