Techniques: Molecular Cloning
Background
In principle, cloning in plasmid vectors is very straightforward. The plasmid DNA is
cleaved with a restriction enzyme and joined in vitro to foreign DNA. The resulting
recombinant plasmids are then used to transform bacteria (see the figure below). In
practice, however, the plasmid vector must be carefully chosen to minimize the effort
required to identify and characterize recombinants.
The major difficulty is distinguishing between plasmids that contain inserted foreign
DNA and vector molecules that have re-circularized without insertion of foreign DNA.
Re-circularization of the vector can be limited to some extent by adjusting the
concentrations of the foreign DNA and vector DNA in the ligation reaction, and by
inclusion of Calf Intestinal Phosphatase (CIP) to the vector species.
CIP, a dimeric glycoprotein, is the enzyme of choice to remove 5'-phosphate groups from
linear DNAs (the generic reaction alkaline phosphatase enzyme catalyzes is shown
below). Because each monomer of CIP carries two atoms of zinc that are essential for the
activity of the enzyme, the buffer used for dephosphorylation should contain ZnCl2 at a
final concentration of 1 mM. At the end of the dephosphorylation reaction, CIP is
removed by digestion with a small amount of Proteinase K in the presence of EDTA, and
the dephosphorylated DNA is then purified by ethanol precipitation.
The ligation of a segment of foreign DNA to a linearized plasmid vector involves the
formation of new bonds between phosphate residues located at the 5' termini of doublestranded DNA and adjacent 3'-hydroxyl moieties. When both strands of the plasmid
vector carry 5'-phosphate residues, four new phosphodiester bonds are generated.
However, when the plasmid DNA has been dephosphorylated, only two new
phosphodiester bonds can be formed. In this case, the resulting hybrid molecules carry
two single-strand nicks that are repaired after the hybrids have been introduced into
competent bacteria (this can be clearly seen in the figure on the first page of this handout;
look at the ligated species prior to transformation into E. coli – see the two ‘nicks’ in the
DNA)?
The formation of phosphodiester bonds between adjacent 5'-phosphate and 3'-hydroxyl
residues can be catalyzed in vitro by two different DNA ligases; E. coli DNA ligase and
bacteriophage T4 DNA ligase (the ligation reaction is shown in the figure below). For
virtually all cloning purposes, bacteriophage T4 DNA ligase is the enzyme of choice
because it will join blunt-ended DNA fragments efficiently under normal reaction
conditions. As far as concentration dependence is concerned, if the concentration of DNA
in the ligation reaction is increased (i.e. much more insert to vector DNA), it is more
likely that a given end of insert DNA will encounter a terminus located on the vector
DNA before intramolecular ligation occurs.
Distinguishing between plasmids containing inserted foreign DNA and recircularized
vector molecules is accomplished with a manipulation of the lacZ gene at the insertion
site of the vector (our vector of interest is pUC19). The galactoside IPTG (Isopropyl-1thio--D-galactoside), which induces the lacZ gene and inhibits the lac repressor protein,
and X-gal (5-Bromo-4-chloro-3-indolyl--D-galactoside), a non-inducing chromogenic
substrate of -galactosidase, are included in the growing medium of the transformed cells
with the appropriate antibiotic (ampicillin). The plasmids produce the -peptide of galactosidase, which complements the lacZ deletion mutation in E. coli strains such as
DH-5. The chromogenic reaction is demonstrated in the figure below.
When such mutant strains are transformed by nonrecombinant vectors, blue colonies are
generated on plates containing ampicillin and X-gal; vectors with inserts give rise to
colorless colonies. In other words, plasmids containing inserted foreign DNA will
inactivate the lacZ gene by inserting at a site within the gene, and hence, colonies from
bacteria containing this recombinant plasmid will not utilize X-gal as a substrate and will
have a colorless appearance. Conversely, colonies containing recircularized vector will
grow blue color colonies, since the lac-Z gene is operational and these bacterium use Xgal as substrate. In reality, the colorless appearance is slightly blue, but relative to the
blue appearance of the non-recombinant species, is easily selected by the naked eye.
A special note about our ligation: directional cloning
Several strategies are available to ligate fragments of foreign DNA to plasmid vectors.
The choice among them depends on the nature of the termini of the foreign DNA
fragment and the nature of the restriction sites in the plasmid vector and the foreign
DNA.
The easiest fragments to clone carry non-complementary protruding termini generated by
digestion with two different restriction enzymes. Most plasmid vectors contain
polycloning sites that consist of recognition sequences for several different restriction
enzymes (you can see this “hi-lited” in the figure below). Given the large variety of
polycloning sites, it is almost always possible to find a vector carrying restriction sites
that are compatible with the termini of the fragment of foreign DNA. The fragment of
foreign DNA is then inserted into the vector by a process known as directional cloning.
For example, the vector pUC19 can be digested with EcoRI and BamHI, and after
digestion, the large fragment of the vector is purified from the small remnant of the
polycloning site by alcohol precipitation (we will forego the physical chemistry here; the
very small remnant DNA fragment does not precipitate very well under these
circumstances, so it is easy to purify the large fragment of the vector).
The vector DNA can then be ligated to a segment of foreign DNA that contains cohesive
termini (overhanging fragments) compatible with those generated by EcoRI or BamHI.
In our case, the PCR amplified GFP gene we have produced was generated using DNA
primers encoding a terminal EcoRI or BamHI recognition sequences. Thus, we are able
to digest the amplified GFP gene we have produced with these restriction enzymes and
produce a directionally digested product for cloning upon purification by alcohol
precipitation (the same explanation applies here as it did for the vector above – we do not
precipitate the smaller remnant DNA fragments produced by the insert digest).
The resulting circular recombinant is then used to transform E. coli to ampicillin
resistance (the vector encodes the -lactamase enzyme, which degrades the drug
ampicillin). Because of the lack of complementarity between the EcoRI and BamHI
protruding ends, the vector fragment cannot re-circularize efficiently and transforms E.
coli very poorly. Therefore, almost all bacterial cells resistant to ampicillin contain
recombinant plasmids that carry the GPF gene forming a bridge between the EcoRI and
BamHI sites. Importantly, the gene is aligned in the correct orientation and due to its
“directional cloning” will produce a functional protein. Of course, different combinations
of enzymes can be used depending on the particular segment of foreign DNA and the
specifications therein.
Purpose
The purpose of this exercise is to familiarize you with the molecular cloning of a gene
into an expression vector. You will clone the polymerase chain reaction amplified gene
for Green Fluorescent Protein into the expression vector pUC19.
Materials per team
(Cloning day 2 – your instructor will perform this procedure prior to Cloning day 3 and will
explain this step to you on Cloning day 3)
1.5 mL Microfuge Tubes
Microfuge Tube Rack
P20 Pipetman
PCR amplified GFP DNA
Pipetman Tips (yellow)
P200 Pipetman
Sodium Acetate
95% Ethanol (cold)
70% Ethanol
Freezer Block (or ice in beaker) Mini and Tabletop Centrifuges 37oC Incubator
BamHI Restriction Enzyme
Purified pUC19 DNA
Deionized Water
EcoRI Restriction Enzyme
10X Restriction Enzyme Buffer Proteinase K Solution
Forceps
(Cloning day 3)
Digested pUC19 DNA
Digested GFP DNA
P20 pipetman
Forceps
95% Ethanol (cold)
P200 pipetman
Freezer Block (or ice in beaker) Mini and Tabletop Centrifuges
Deionized Water
Proteinase K solution
10X CIP Dephosphorylation buffer
Microfuge Tube Rack
Pipetman tips (yellow)
Sodium Acetate
70% Ethanol
37oC Incubator
CIP enzyme
(Cloning day 4)
Dephosphorylated and digested pUC19 DNA
Purified and digested GFP DNA
P20 pipetman
Forceps
Pipetman tips (yellow)
95% Ethanol (cold)
P200 pipetman
Sodium Acetate
Freezer Block (or ice in beaker) Mini and Tabletop Centrifuges 70% Ethanol
Deionized Water
37oC Incubator
Microfuge Tube Rack
10X T4 DNA ligase buffer
T4 DNA ligase enzyme
10 mM ATP
1.5 mL Microfuge Tube
Procedures
(Cloning day 1) – This period has already been performed by you; the PCR
amplification of the GFP gene from jellyfish chromosomal DNA.
(Cloning day 2) – This period starts off with an ethanol purification of your PCR
amplified GFP DNA sample from day 1. After that, you will enzymatically digest
the vector DNA (pUC19) and the insert DNA (GFP). Your instructor performs this
step for you.
1. Grab a 1.5 mL microfuge tube and a microfuge tube rack. Also obtain your PCR
amplified GFP DNA sample (from last period). It has been stored in the -20oC
freezer.
2. Purify your PCR amplified GFP DNA in a 1.5 mL microfuge tube by mixing the
following reagents as follows (label the tube “GFP”):
PCR amplified GFP (DNA sample)
3M Sodium Acetate, pH 7.0
95% Ethanol
Total Volume
50 L
5 L
100 L
~155 L
3.
4.
5.
6.
Mix the tube by gentle tapping with your finger (the instructor will show you how).
Store the tube on ice (or a freezer block) for 15 minutes.
Centrifuge the tube for 10 minutes at maximum velocity (16K rpm).
Decant the supernatant (liquid) from the microfuge tube (use a P200 micropipet).
When you pipet off the volume, draw the liquid from the opposite side of the tube
that the DNA would pellet on (ask your instructor what this means before you act).
7. Wash the sample with 200 L of 70% ethanol.
8. Centrifuge the tube for 1 minute at maximum velocity (16K rpm).
9. Decant the supernatant (liquid) from the microfuge tube (once again, successively
draw off the volume with a P200 micropipet).
10. Air-dry the sample in the hood (about 20 minutes) or a preheated PCR block (5 minutes).
11. Next, you will produce two, double enzymatic digestions in two, separate 1.5 mL
tubes. One digestion is for the vector DNA, pUC19, which is already purified by the
biotechnology company (supplier). The other digestion is for the insert DNA, GFP,
which was PCR amplified and purified by you in the previous steps above. Of
course, the GFP DNA will be directionally cloned (in the correct orientation) into
pUC19 at the polylinker cloning site. But first, we have to resuspend the purified
GFP (insert DNA) in water. At present, the sample is lyophilized (freeze dried).
12. Add 20 L of water to the purified GFP DNA sample.
13. From your instructor, obtain a 1.5 mL microfuge tube containing a 20 L pUC19
DNA sample from your instructor. The tube is labeled “pUC.”
14. Independently, double-digest insert GFP DNA or vector pUC19 DNA with both
restriction enzymes (BamHI and EcoRI) as follows:
Resuspended DNA (GFP or pUC19) in H2O
H2O
10X Reaction Buffer
Enzymes (1 L aliquot of each enzyme)
Total Volume
15. Mix the tubes by gentle tapping with your finger.
16. Spin down the samples in a portable microfuge for 5 seconds.
17. Place the samples in the rack in the incubator for 1 hour at 37oC.
18. Add 5 L of Proteinase K solution to your enzymatic reaction.
20 L
5 L
3 L
2 L
~30 L
19. Mix the tubes by gentle tapping with your finger. Place the samples in the rack in
the incubator for 30 minutes at 37oC.
20. Store the samples at -20oC until further use.
(Cloning day 3) – This period starts off with an ethanol purification of your digested
samples from day 2. After that, you will enzymatically dephosphorylate the vector
DNA (pUC19).
1. Grab a microfuge tube rack. Also obtain your restriction digested pUC and GFP
DNA samples from last period (put the samples in the rack). They have been stored
in the -20oC freezer.
2. Independently purify your DNA samples by mixing the following reagents as follows
(you have two tubes of samples labeled GFP or pUC19; you will do the following
purifications in these tubes):
Restriction Digested DNA (GFP or pUC19) in H2O
35 L
3M Sodium Acetate, pH 7.0
3 L
95% Ethanol
60 L
Total Volume
~100 L
3.
4.
5.
6.
Mix the tube by gentle tapping with your finger (the instructor will show you how).
Store the tube on ice (or a freezer block) for 15 minutes.
Centrifuge the tube for 10 minutes at maximum velocity (16K rpm).
Decant the supernatant (liquid) from the microfuge tube (use a P200 micropipet).
When you pipet off the volume, draw the liquid from the opposite side of the tube
that the DNA would pellet on (ask your instructor what this means before you act).
7. Wash the sample with 200 L of 70% ethanol.
8. Centrifuge the tube for 1 minute at maximum velocity (16K rpm).
9. Decant the supernatant (liquid) from the microfuge tube (once again, successively
draw off the volume with a P200 micropipet).
10. Air-dry the sample in the hood (about 20 minutes) or a preheated PCR block (5
minutes).
11. Give your GFP (insert DNA) sample to your instructor. It will be stored at -20oC
until next period.
12. Next, you will produce an enzymatic dephosphoryation of pUC19, your vector
DNA.
13. Resuspend the lyophilized (freeze dried) pUC DNA sample with 90 L of H2O.
14. Add 10 L of 10X CIP dephosphorylation buffer to the sample.
15. Add 1 L of Calf Intestinal Phosphatase (CIP) enzyme to the sample.
16. Mix the tubes by gentle tapping with your finger.
17. Spin down the samples in a portable microfuge for 5 seconds.
18. Place the samples in the rack in the incubator for 30 minutes at 37oC.
19. Add 5 L of Proteinase K solution to your enzymatic reaction.
20. Mix the tubes by gentle tapping with your finger. Place the samples in the rack in
the incubator for 30 minutes at 37oC.
21. Store the samples at -20oC until further use.
(Cloning day 4) – This period starts off with an ethanol purification of your
dephosphorylated and digested pUC19 DNA sample from day 3. After that, you will
enzymatically ligate the dephosphoryated and digested vector DNA (pUC19) to the
digested insert DNA (GFP).
1. Grab a microfuge tube rack. Also obtain your dephosphoryated and restriction
digested pUC19 DNA sample, and your lyophilized (freeze dried) restriction
digested GFP sample from last period (put the samples in the rack). They have been
stored in the -20oC freezer.
2. Independently purify the dephosphoryated and restriction digested pUC19 DNA sample
by mixing the following reagents as follows:
dephosphoryated and restriction digested pUC19 DNA sample in H2O 106 L
3M Sodium Acetate, pH 7.0
9 L
95% Ethanol
180 L
Total Volume
~300 L
3.
4.
5.
6.
Mix the tube by gentle tapping with your finger (the instructor will show you how).
Store the tube on ice (or a freezer block) for 15 minutes.
Centrifuge the tube for 10 minutes at maximum velocity (16K rpm).
Decant the supernatant (liquid) from the microfuge tube (use a P200 micropipet).
When you pipet off the volume, draw the liquid from the opposite side of the tube
that the DNA would pellet on (ask your instructor what this means before you act).
7. Wash the sample with 200 L of 70% ethanol.
8. Centrifuge the tube for 1 minute at maximum velocity (16K rpm).
9. Decant the supernatant (liquid) from the microfuge tube (once again, successively
draw off the volume with a P200 micropipet).
10. Air-dry the sample in the hood (about 20 minutes) or a preheated PCR block (5 minutes).
11. Next, you will produce an enzymatic ligation of the purified, lyophilized (freeze
dried) dephosphoryated and digested vector DNA (pUC19) to the purified
lyophilized (freeze dried), digested insert DNA (GFP).
12. I am not certain how much of both purified DNA species you have, so we will have
to take a “rough guess” and hope the Gods of Science favor us. We started this
experiment with twice the concentration (in weight, or g) of insert DNA (GFP) to
vector DNA (pUC19) species. Certainly, we have lost some DNA during
purification (approximate 50% loss), but since we performed the purification
procedures on both species of DNA, the scale of lost DNA should be the same for
each species (this makes sense, right). Starting concentration of GFP: 2g. Starting
concentration of pUC19: 1g. After purification (50% lost), what are the final
concentrations of the digested insert DNA (GFP) and the digested, dephosphorylated
vector DNA species (pUC19)?
13. Next calculation… Generally, you want an insert to vector DNA molar ratio of 3:1
during cloning (a great excess of insert DNA to vector DNA). How would you
calculate this if your insert DNA is ~1 kb in length (1kb/mol) and your vector DNA
is ~3 kb in length (3kb/mol)? I see smoke coming from your ears; discussion time!
14. Having calculated your variables, resuspend the lyophilized (freeze dried)
dephosphoryated and digested vector DNA (pUC19) sample and the digested insert
DNA (GFP) sample with 20 L of deionized H2O each.
15. Resuspend the invisible pellet of DNA well (remember where on the bottom of the
tube it is located). Mix the tube by gentle tapping with your finger (the instructor
will show you how) and pulse-spin down.
16. Label a fresh, sterile 1.5 mL microfuge tube. Aliquot 10 L of dephosphoryated and
digested vector DNA (pUC19), and 5 L of digested insert DNA (GFP) to the tube.
17. Incubate the mixed DNA solution tube at 37oC for 5 minutes. (Place in the rack in the
incubator)
18. Chill the mixed DNA solution tube on ice or in a freezer block.
19. Process the ligation by adding the following reagents to the mixed DNA solution tube:
Processed GFP DNA
5 L
Processed pUC19 DNA
10 L
(DNA is already in the tube…)
10X T4 DNA ligase buffer
T4 DNA ligase enzyme
10mM ATP
Total Volume
2 L
1 L
2 L
20 L
20. Mix the tube by gentle tapping with your finger and pulse-spin down.
21. Incubate the reaction at room temperature overnight (give your sample to your
instructor). The sample will be stored at -20oC until the next lab period, when you
perform the bacterial transformation and generate a recombinant organism (now who
is playing God…).
WORKSHEET
Molecular Cloning
1. In summary, what are the five procedural steps you will take to clone GFP into a
bacterium (look at the first figure of the exercise for the answer)?
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2. What are two ways you can limit the extent of re-circularization of the vector during
ligation?
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3. Would you consider Alkaline Phosphatase to be a Fibrous or Globular protein? Why
(the structure of a protein belies its function)?
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4. Why is Calcium necessary for the phosphatase reaction to proceed? Why employ
Proteinase K and EDTA after the reaction has been completed?
(Hint: http://wiki.answers.com/Q/What_the_function_of_EDTA_during_DNA_extraction)
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5. Alkaline Phosphatase catalyzes what kind of reaction (hydrolysis or dehydration)?
6. DNA ligase catalyzes the formation of a phosphodiester bond between adjacent 5’phosphate and 3’-hydroxyl residues. Why is it so important to have an excess molar
concentration of insert to vector DNA during the ligation reaction?
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7. Looking at the figure depicting DNA ligation in your exercise (it is on the third page,
the cartoon below is just for fun-and-giggles), ________ is required for the reaction to
proceed in eukaryotes. Why?
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(More about nucleophilic attack at: http://en.wikipedia.org/wiki/Nucleophilic_substitution)
8. What is the significance of including IPTG in the growth media when distinguishing
between bacterium containing plasmids composed of inserted foreign DNA or
recircularized vector molecules.
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9. Vectors containing insert DNA will give rise to (blue or colorless) colonies? Why?
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10. What is a polylinker cloning site in a vector? What is its significance in cloning?
Where are the polylinker cloning sites located in pUC19 (look at the vector map in
the figure in the exercise)?
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11. What is -lactamase and what does it do? Why is it an important part of the parent
vector pUC19?
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12. What is directional cloning? What is the importance of compatible, cohesive termini
generated by DNA digestion to this process (Hint: EcoRI and BamHI)? How does
this contribute to the product being produced?
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