Reading Assignment

Working with DNA, pp. 11-146. (On Library Reserve)

Chapter 2 The recombinant DNA laboratory
2.1 Supplies 11
2.2 Reagents 17
2.3 Technique 24
2.4 Looking at DNA on a gel 35
Biology 572 2.5 Safety 49
2.6 Documentation 58
2.7 Special lab problems 61
Solutions to problems 66
Chapter 3 Solutions, buffers, stocks and cocktails
3.1 Solutions, suspensions and slurries 67
Recombinant DNA Techniques 3.2 Solubility of macromolecules 69
3.3 Solute concentration 72
3.4 Solution pH 75
3.5 Precipitation methods 76
3.6 Preparing a solution 81
3.7 Buffer considerations 88
3.8 Stock solutions 100
Solutions to problems 105
Chapter 4 Cloning vectors
4.1 Plasmids 109

Metric units and prefixes Making solutions

What is a “solution”?
grams moles liters What does “concentration” of a solute mean?
Milli (10 -3 ) mg mmole ml What is the right way to make a solution?
Micro (10 -6 ) µg µmole µl
Nano (10 -9 ) ng nmole 1000 cm 3 1 liter
Pico (10-12 ) pg pmole
Femto (10 -15 ) fg fmole

For example: 100 pg = 0.1 ng

Why is concentration important?
…and 10,000 µl = 10 ml … things bump into each other
in solution!

Solution problems Solution problems

1. The formula weight of NaCl is 58.44. How many grams of 2. What would be the molarity of the same solution if you
NaCl would you weigh out if you wanted to make 100 ml of prepared it in 20 ml instead of 100 ml?
a 0.1 M solution?

1 liter 0.1 moles 58.44 g =?

100 ml X X X 1 mole Think about the concept of concentration - as a proportionality:
1000 ml 1 liter
C = solute/volume

If the denominator is decreased by a factor of 5, what happens

to the value of C?
More problems of this kind on p. 88

1
 Solution problems
Sodium acetate has a FW of 82.03
3. How many grams of sodium acetate would you weigh out
if you wanted to make 100 ml of a 1 M solution?
4. What would be the molarity of the same solution if you
prepared it in 200 ml instead of 100 ml?
Exactly what steps would you take to make this
solution correctly?
Molarity and % w/v
If you had 50 ml of a solution that is 154 mM NaCl (FW = 58.4),
also known as a “standard saline” solution, how would you
calculate its concentration of NaCl as % (w/v)?
A comment about units …
If a solute has a concentration of 10 mM (milliMolar)
…then these all mean the same thing:
The concentration is: 10 mmole/l
10 µmole/ml
10 nmole/µl
How many pmoles of solute would there be in 5 µl of the solution?
Using stocks to make dilutions
Suppose that you’ve prepared these three stock solutions:
1 M NaOH (FW = 40.0)
1 M NaCl (FW = 58.4)
10% (w/v) SDS sodium dodecyl (or lauryl) sulfate
Using these stocks, how (exactly) would you prepare these?
50 ml of a solution that is 154 mM NaCl?
10 ml of a solution that is 0.2 M NaOH, 1% SDS
Molarity and density
Acetic acid is a liquid, and in its pure (glacial) form it has a
density (at 25°C) of 1.05g/ml.
The FW of acetic acid is 60.05.
What is the molarity of glacial acetic acid?
Solutions prepared using
hydrated vs. anhydrous chemicals
1. How many grams of MgCl2 • 6H2O (FW 203.3) would you
weigh out if you wanted to make 100 ml of a 0.5 M solution?
2. How many grams of anhydrous MgCl2 (FW 95.21) would
you weigh out if you wanted to make 100 ml of a 0.5 M
solution?
Why do we want hexahydrate vs. anhydrous reagent? Do you
compensate by adding less water in the first case?
2
 What are pH buffers? Example of acid/base pair
They’re made up of conjugate base and acid forms.

• What is the Brønsted acid/base pair for a

H+ carboxylic acid (e.g. acetic acid)?
H+

H+
pKa = 4.75 (acetate)
H+
Acid form Base form
http://course1.winona.edu/ sberg/ChemStructures/ Dissocia.gif

Different buffering substances

have different pKa values Example of acid/base pair
H+
H+ • What is the Brønsted acid/base pair for an
H+ amine (e.g. Tris (hydroxymethyl) aminomethane)?
H+ Acid form Base form
pKa = 8
H+
+
H+ H+ NH3

H+ H+
H2O
H+ H3O+
http://olw. mit.edu/media/glossary/tris.jpg
Read section 3.7 carefully

The Henderson-Hasselbalch Setting pH (one way)

Formula Add some acid form

pH = pKa - log10 ([acid form]/[base form]) [acid form]

pH = pKa - log10
• pKa is the negative log of the acid dissociation constant - it is
[base form]
characteristic of a buffering species, although it may vary with
temperature
• We can set the pH of a buffered solution by controlling the ratio
of acid to base form.
• The pH of a solution is associated with a specific ratio of acid to …and separately, some base form
base form.

3
 Setting pH (another way) How do we make 50 ml of a
2. Add a strong acid
to convert some of
5 M KOAc buffer (pH = pKa)?
the base form Need: 2.5 M x 0.05 liter = 0.125 moles KOAc
(FW 98.14) It’s a buffer!
pKa = 4.75
[acid form]
pH = pKa - log10
[base form] Protons
convert
mix

1. Add all the buffer

species as base form
0.25 moles/0.05 liters
Need: 2.5 M x 0.05 liter = 0.125 moles HOAc =5M
(17.5 M acetic acid)

A problem using buffers Bring together two linear equations

The formula weights of Tris base and Tris-HCl are 121.1 and
1. [base form] + [acid form] = total concentration
157.6, respectively, and the pKa of Tris is 8.2. If you combine
1.211 g of Tris base and 1.576 g of Tris-HCl in a 100 ml
2. [acid form]/[base form] = 10 -(pH-pKa)
solution, what is the molar ratio of the acid form and base
form of Tris? What is the pH of the solution?

How would you make a buffer of the same concentration,

but with a pH of 7.2?
(#2 comes from Henderson-Hasselbalch)
See p. 91, and more problems like this on p. 99 pH = pKa - log10 ([acid form]/[base form])

Controlling the ratios (directly) Controlling the ratios (indirectly)

• If you add 6.05 g of Tris base (FW 121.1) to a solution, then decide
that you want there to be an acid:base molar ratio of 2.0, how much
Tris-HCl (FW 157.6) would you add to the solution?
• How many total moles of Tris (both acid and base) would now be
present in the solution?
• If you add 12.11 g of Tris base (FW 121.1, pKa 8.0) to a solution, then
add enough HCl to the solution so that the pH is 8.0, what would be
the molar ratio of the forms in the Brønsted acid/base pair?
• How many total moles of Tris (both acid and base) would now be
present in the solution?

4
 We can prepare a good buffer
Adding NaOH to Tris acid?
within about 1 pH unit of the pKa
• If you prepare a 1 l solution with 15.76 g of Tris-HCl (FW 157.6) and For example, suppose we want to prepare 100 ml of
enough NaOH so that the pH is 8.0, what would be the molar ratio of 1M Tris buffer at pH 7.8
the forms in the Brønsted acid/base pair? pH = pKa - log(acid/base)
• What concentration of Tris-Cl do you write on the label of the bottle?
• Is this the same as a solution of the same concentration and pH
7.8 = 8.2 - log(acid/base)
prepared with Tris base and HCl? log(acid/base) = 0.4
acid/base = 100.4 = 2.51
…also acid + base = 0.1 mole (total)
You could say, the 0.1 moles = 2.51 + 1 “parts” = 3.51 “parts”
… so 1 “part” = 0.1/3.51 = 0.0285 mole
(that’s the base form we need, and the acid form is 0.1-0.0285 = 0.0715 mole)

How do we check our Concept: the acid/base ratio is

calculation? most stable at 1:1
• The general concept: When you add a small amount of strong acid to a
1. Check the total concentration: buffer, you add to the numerator (the “acid form”) and subtract from
acid form + base form = total the denominator (the “base form”) of the acid/base ratio. Which of
these ratios is changed least, as x increases from 0 to (say) 10?
0.0715 mole + 0.0285 mole = 0.1 mole (total)
(909+x)/(91-x) (near 10:1 ratio)
(500+x)/(500-x) (near 1:1 ratio)
2. Check the ratio and pH: (91+x)/(909-x) (near 1:10 ratio)
pH = 8.2 - log(acid/base)
= 8.2 - log (0.0715 mole / 0.0285 mole )
= 8.2 - log (2.51)
= 8.2 - 0.4
= 7.8

Calculation of buffer behavior Calculation of buffer behavior

Suppose you’ve got 100 ml of a 1 M Tris-Cl buffer (pKa
8.2) at a pH of 10.2. How does its pH change if you add 50 Step 2: Bring in the overall concentration equation to
µl (about 1 drop) of concentrated (12.4 M) HCl? determine the number of moles of each form.
acid + base = (0.1 l) (1 mole/l) = 0.1 moles
Step 1: Calculate the acid base ratio from the pH
or acid = 0.1 moles - base
pH = pKa - log(acid/base) Remember acid/base = 0.01, so
(0.1 - base)/base = 0.01
10.2 = 8.2 - log(acid/base)
0.1 - base = 0.01 base
so log(acid/base) = -2 0.1 = 1.01 base
and (acid/base) = 10-2 = 0.01 …finally base = 0.1/1.01 = 0.0990 moles
and acid = 0.1 - 0.0990 = 0.000990 moles

5
 Calculation of buffer behavior Calculation of buffer behavior
Step 3: Figure out how much acid you’ve added, and what Step 4: Now that you’ve got a new ratio, what is the new
effect it has on the ratio of the buffer forms pH associated with that ratio?
The one drop of HCl has: acid/base = 0.0984/0.00161 = 0.01513
50 µl (1 liter/10 6 µl) (12.4 moles/liter) = pH = pKa - log(acid/base)
0.000620 moles HCl = 8.2 - log(0.01513)
= 8.2 - -1.82
After addition: = 10.02
Acid form = 0.000990 moles + 0.000620 moles = 0.0984
Base form = 0.0990 moles - 0.000620 moles = 0.00161 The main idea: The pH of the 100 ml of 1 M Tris-Cl dropped
from 10.2 to 9.99, after addition of a drop of concentrated HCl
The main idea: With the addition of a small amount of strong
acid, we protonate some of the free base, converting it to the
acid form.

Calculation of buffer behavior Calculation of buffer behavior

How does the concentration of the buffer affect the How does the initial pH of the buffer affect the calculation?
calculation? Keeping the volume at 100 ml, here are some Keeping the volume at 100 ml and the buffer concentration
results from adding the one drop of HCl: at 1 M, here are some results from adding the one drop of
HCl:
1 M Tris-Cl 10.2 10.0 1 M Tris-Cl 10.2 10.0 (drop of 0.20)
0.5 M Tris-Cl 10.2 9.89 1 M Tris-Cl 9.2 9.17 (drop of 0.026)
0.1 M Tris-Cl 10.2 9.40 1 M Tris-Cl 8.2 8.19 (drop of 0.009)
0.05 M Tris-Cl 10.2 9.11 1 M Tris-Cl 7.2 7.17 (drop of 0.027)
0.01 M Tris-Cl 10.2 8.18 1 M Tris-Cl 6.2 5.89 (drop of 0.308)

The main idea: The strength of the buffer affects how well it can The main idea: The strength of the buffer affects how well it can
arrest the drop in pH. arrest the drop in pH.
Reality check: our 0.000620 moles HCl would be 0.0062 M in Reality check: our 0.000620 moles HCl would be 0.0062 M in
water, which would give us a pH of 2.21 if there were no Tris-Cl water, which would give us a pH of 2.21 if there were no Tris-Cl

The titration curve shows

pH stability depends on pH Conceptual problems
100 ml of 0.1 M • Tris is a short name for Tris (hydroxymethyl)
Tris-Cl, pH 9.2 aminomethane, and the functional group that makes it a
buffering compound is the amine (pKa 8.0). Would Tris
… titrated with be mostly positively charged, negatively charged, or
1 M HCl uncharged at pH 6.0, 8.0, and 10.0?
• Suppose you have a potassium acetate solution. Would the
acetate be mostly positively charged, negatively charged,
or uncharged at pH 2.0, 5.0, and 8.0?
• Glycine has an amino group (pKa 9.6) and a carboxylic
acid group (pKa 2.34). How would glycine be charged (or
not) at pH 1.0, 7.0, and 12?

6
 Conceptual problems EDTA solutions
• Has four pKa values: 1.99, 2.67, 6.16 and 10.26
• If you prepared a solution of sodium acetate
• How would you prepare 0.5 M trisodium EDTA (at approx. pH 8.0),
salt, would you add a strong acid or base to starting from EDTA free acid and NaOH?
bring it to its pKa? • How would you prepare 0.5 M trisodium EDTA by combining EDTA
free acid and tetrasodium EDTA?
• If you prepared a solution of Tris base,
would you add a strong acid or base to bring
it to its pKa? Trisodium is soluble…

http://www.fishersci.com/wps/portal/PRODUCTDETAIL?productId=604
212&catalogId=29104&pos=2&catCode=RE_SC&fromCat=yes&keepSe
ssionSearchOutPut=true&brCategoryId=null&hlpi=y&fromSearch=Y

Example: The Example:

amino acid lysine aspartic acid
•For what pH range is this •For what pH range is this
representation of charges representation of charges
generally correct? generally correct?
•How would you describe the •How would you describe the
charge distribution of lysine at charge distribution of aspartic
pH 1, 4, 7, 9, or 12? acid at pH 1, 4, 7, 9, or 12?
•If you had a reagent bottle •If you had a reagent bottle
marked simply L-lysine (i.e. marked simply L-aspartic acid
not a salt), and make a solution (i.e. not a salt), and make a
from its crystals, exactly what solution from its crystals,
have you got, and what is the exactly what have you got, and
approximate pH? what is the approximate pH?
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Graphics/ChemStructSM/Lys.gif http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Graphics/ChemStructSM/Asp.gif

There is a pH where these have no

net charge

50% dissociated
at pH 9.47
(possibly an
50% dissociated
at pH 2.98 (possibly
DNA
empirical finding) an empirical finding)

They can be crystallized as zwitterions - they are their own counterion

Clinical Chemistry (M.L. Bishop, E.P. Fody, L.E. Schoeff) p. 189
http://books.google.com/books?id=ee2rOklb0qwC&pg=PA189&lpg=PA189&dq=%22solution+of+aspartic+acid+has+a+pH%22&source=web&ots=4jl1XTiZ
E7&sig=0Hosh7iZiA_0VETHpNIrcgzXmHk&hl=en&sa=X&oi=book_result&resnum=6&ct=result#PPA189,M1

7
 The sugar in DNA is deoxyribose Polymerization is 5’ to 3’
O
|
Base O - P=O Base
|
5ʼ

5ʼ

O O O
| | |
O=P - O - P - O - P=O Base
|| || |
O O 5ʼ

3ʼ

3ʼ

dsDNA is anti-parallel
Key elements of plasmids we use
5’ phosphate Origin of DNA replication
- for extrachromosomal replication

3’ hydroxyl
Selectable marker gene
- for stable maintenance

Plasmid maintenance (?) Plasmid maintenance (?)

In the PRESENCE of antibiotic… In the ABSENCE of antibiotic…

+ + , ,

Plasmid-carrying cell Plasmid-free cell stops Plasmid-carrying cell Plasmid-free cell also
survives and propagates growing survives and propagates survives and propagates

8
 Foreign genes can be expressed However, some foreign genes can
from plasmids be toxic to a cell when expressed!
protein protein

RNA RNA

DNA DNA

www.lcc.ukf.net

A single “nick” will relax a

DNA supercoiling
supercoiled circle

nick

supercoiled relaxed
http://web.archive.org/web/20000824031912/http://phage.bocklabs.wisc.edu/0014b.jpg

A relaxed circle may have several A relaxed circle may have several
nicks nicks

What type of bonds keep What happens if two

this circle together? nicks on opposite strands
are very close to each
other?
If we put this DNA in
boiling water, how
many single stranded
pieces would we
relaxed release? relaxed

9
 Solubility considerations
DNA in solution
Na+
Na+
+
_ Na
Na+ _ _ Na+
Na+_ _ _
_ _
_ _ Na+ _ _ Na+
_ Na+ _
_ Na+ _
Na+ _ _ _ _ Na
+

_ Na+ Na+
_ Na+
Na+
_ _ Na+
High ionic _ _
Low ionic _ _
strength +
Na_ _ Na+ Na+ _ _ Na
+
strength _ _
_ _ Na+
_ Na+_
_
Na+ _ Na+
Na+
_
Why the change in shape? DNA is a polyanion. Na+ Na+
Na+, K +, Li +, Mg2+ provide cationic shielding of the phosphates
Electrostatic repulsion prevents aggregation and precipitation
pp. 67-72

Effects of electrostatic shielding Can DNA become uncharged at low pH?

Li+ Li+ Cl- _
- - + Na+ Li+ - H+ _ H+
Cl-+ Cl Na+ Li+ Cl+ Na Li+ + Cl
_ _Li Na+ Li+ _
Li Na+ _ _ Li Cl- Na+ H+ _ H+
Cl- Li+
_ _ + + Li+ _
_ _ Li+ Cl- _ _ H+
_ _ Li Na Cl- + _ Na+ H+ _
Cl- +
Li_ _ Li+ Na+ NaLi+ _ H+ _ H+
_ Li+ Cl+ - H+ _ _ H+
Na+ _ _ Cl- _ _ _ LiNa+ Cl- _
Cl Li _
- + Cl - Na+
_ +Na+ Cl- NaNa+ _ + Li+
H+ _ H+
Li+ Li
_ Cl- Na+ H _
+
_ H+
+ _ _ Li+_ Cl- Cl- +Li+ _ _ Cl- Li++ _ _ H+
Cl- Li Na+ _ Na _ _ _ Na Cl- H+
_ _ _ + Na+ -Cl- Li+ _ H+ _ _ H+
Cl- Li Cl _ Li++ Cl-
_
Cl- Na+ _ _ Li+
Na
H+ _
+ Cl-
_ H+
Li+
Na+ LiCl
- Li+ Cl- + Cl- Na H+
_ +
Na+ Li-
+
Cl- Na+ Cl- H
Cl- Na + Cl

Decreased repulsion (closer approach), and decreased coil radius. Yes - that’s why we call it a nucleic “acid”
However, the surrounding water decreases this effect because it is polarizable. We add
ethanol or isopropanol to decrease the overall dielectric constant (decrease the polarizability DNA and RNA are more “nonpolar” at pH 4 to 5 than at neutral pH
of the solvent) and this finally causes the aggregation and precipitation.

Examples of charged amino acid side chains:

lys and asp Charges on proteins are also pH dependent

H3O+
0 _ 0 _ + _
_ + 0 + 0 +
_ 0 0
+
_ +
_ +
_
_ _ 0
0 _ + + _ + + _ +

Net -3 charge Net 0 charge Net +2 charge

at pH 8.5 at pH 7.5 at pH 6.5

The pH at which a protein has net zero

http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Graphics/ChemStructSM/Lys.gif
charge is called the “isoelectric point” or pI
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Graphics/ChemStructSM/Asp.gif

10
 … it may take an increase in ionic strength to get a
We’re feeling less protein to precipitate, if there is remaining
A protein is usually least self-repulsion than electrostatic repulsion.
soluble at its pI at other pH values!
Na+
Cl-
Li+ Cl- 0 _
Li+ 0
0 _ 0 _ 0 0 _ +
0 0
_ Cl- 0
+ 0 _ + 0 + + 0
+ Li+
0 0 + 0 0 0 _
+
_ +
_ +
_ +
_ _ Cl-
0
_ +
_ _ _ _ + _ +
+ _ + _ + _ + + + _ + 0 Na+
_ + Na+ _ Cl-
+ _ + 0 _ Cl- 0 +
Na+ 0 +
0 _ 0
0 _ +
_ Li+
0 + 0
0 + +
_ _
0 _ +
0 +
_ _ +
+
_ +
_ _ _ +
+ _ +
+ _ + This is called “salting out” a protein…

Conversely, a protein may be aggregated at low ionic

strength if there is electrostatic attraction, and salt
may need to be added to bring it into solution. Gel electrophoresis
Li+ cathode
0 _ Cl- Na+ 0 _
0 + Cl- 0 +
Cl-
Li+ 0
+
_ 0 _ +
_
_ Li+
0 + _ Cl-
+ _ + 0 + _ + DNA
0 +
_
_ migration
+ _ + 0 Na+
Na+ Na+ _ Cl-
Cl- 0 _ 0 +
0 + 0
+
_ Li+
0
+
_ _ anode
_ + _ +
+ Why does DNA run towards red?
_ +
Fig. 2.3A
This is called “salting in” a protein…

Ethidium bromide dye intercalates

Gel electrophoresis between bases when it binds and distorts
the double helix
Rotate
to
relieve
tension

Relationship between migration

and linear DNA size?
Fig. 2.3 http://sandwalk.blogspot.com/2007/07/ethidium-bromide-binds-to-dna.html

11
 What would equimolar amounts
of these fragments look like on a Migration of DNA forms on a gel
gel?

5000
4000
3000
2000

1000

If you loaded 100 fmoles of each fragment, how many ng of The shape and size of the DNA are both important in
DNA would be in each band (see Table 2.1 for help) determining gel migration.

Neutralization and collapse of

Alkaline lysis of E. coli
debris

plasmid
1% SDS (sodium dodecylsulfate)
0.2 N NaOH
ssDNA of
genome 5 M KOAc (pH = 4.75)

plasmid
Separate by centrifugation

Electrophoresis after extraction Alcohol precipitation of DNA

•Salt - for electrostatic shielding

•Ethanol or isopropanol - to decrease
nicked
the dielectric constant of the solution
supercoiled

Residual RNA
http://www.nature.com/nprot/journal/v1/n6/thumbs/nprot.2006.452-F2.jpg

12
 Principles of DNA purification by affinity
resins Ethidium bromide decreases the net
density of DNA when it binds

Adding ethidium
e.g. Promega®
bromide decreases
density
supercoiled
DNA
density

Supercoiled DNA
e.g. Qiagen® reaches a limit in
absorption of ethidium
bromide dye

Fig. 4.1 nicked/linear

Plasmid DNA separation by CsCl

Storage of DNA
density gradient centrifugation
• Dry pellet
• Ethanol precipitate
• Aqueous solution (4ºC, -20ºC, -70ºC) with
10 mM Tris-Cl (pH 7 to 8), 0.5 mM EDTA

Nuclease nibbling can be a problem … if the ends need to

have a specific structure for cloning
Fig 4.2
http://www.nature.com/nature/journal/v403/n6770/images/403646aa.0.jpg

Can DNA be handled too roughly? Plasmid features

• High MW DNA is susceptible to shearing, • Origin of replication
by shaking or vortexing in solution, or – Different copy number per cell (ColE1, F, P1)
pipetting through a narrow bore pipet. – May be unidirectional (e.g. f1, M13) -> ssDNA
• Plasmids smaller than 10 kbp are resistant – Host range of an origin is limited
to this type of damage, but can be broken – Origins in eukaryotes (e.g. EBV oriP, 2µ circle,
into fragments by sonication (high energy ARS elements)
sound waves from a probe) – Foreign DNA may be integrated into the
genome in absence of an ori

13
 Plasmid features Plasmid features
• Selectable marker • Cloning sites
– Antibiotic resistance (e.g. Kan, Amp, Hyg, G418 -host – “Polylinkers” or “multiple cloning sites” (MCS)
cell must be susceptible to drug) – More optional features:
– Complementation of auxotrophic mutation (e.g. URA3 • Transcriptional promoter for expression in cell
in S. cerevisiae) • Phage RNA polymerase promoters for in vitro expression of
– Herbicide resistance (e.g. bar gene for Bialophos® RNA
EcoRI
resistance in plants) • Gateway® (Invitrogen) sites lacZ gene
• Coding sequence interruption
– Expression from appropriate regulatory sequences
– Principle of rescue varies, as does the location of effect
(inside or outside the cell)
insertion
(detection with IPTG (lactose analog) and X-gal (colorimetric substrate))

Still more
Plasmid cloning capacity
options
• ColE1, pUC plasmids
• “Poison sequence” to prevent – e.g. for maintenance of up to 10 kbp, usually at high
propagation of plasmids copy number
lacking an insertion at the
cloning site • BAC, PAC vectors
– e.g. for maintenance of large genomic DNA regions, up
to 250 kbp at low copy number

Fig 4.3

ribosome

A foreign protein
Fusions and tags
can be expressed Glutathione S-transferase (GST) - the fusion binds to
as a “fusion glutathione affixed to a column, and can be released by
protein”, if it is free glutathione
cloned “in frame” 6HIS - the fusion (tag) binds to nickel chelated to a
into an expressed column, and can be released by imidazole
sequence.
FLAG® - the fusion (tag) binds to a monoclonal anti-
FLAG antibody affixed to a column, and can be
released by a pH shock (e.g. 10 mM glycine pH 3.5)

Fig 4.4

14
 Other types of fusions
• GFP - for positional reporting of a product
(where is it expressed or compartmentalized in a cell?)
• Signals for protein secretion in prokaryotes or eukaryotes

Fig 4.5

For ligation using T4 DNA ligase,

Principles of Ligation
the DNA ends must fit together
The DNA ends must:
These two fragments have overhanging ends that fit together by
base pairing like pieces of a puzzle, and they could be ligated
• fit together
together on both the top and bottom strand:
• have a 5’ phosphate and 3’ hydroxyl
• find each other in solution
5’-...GGAGATCAGACTA CGATCAGACAGCT...-3’
3’-...CCTCTAGTCTGA TGCTAGTCTGTCGA...-5’

The product of ligation would have covalently joined strands:

5’-...GGAGATCAGACTACGATCAGACAGCT...-3’
3’-...CCTCTAGTCTGATGCTAGTCTGTCGA...-5’

p. 133

Ends must Ligations generate a mixture of products,

some of which we don’t want
find each
other in
solution
T4 DNA
ligase

This is an issue of DNA concentration

Fig 4.7

15
 5’ phosphate and 3’ hydroxyl are
required for T4 DNA ligase
(removes phosphate group)
Fig 4.6
Introduction of DNA into cells
•Transformation (prokaryotic)
– “competent” cells
– CaCl2 vs. electroporation methods
•Transfection (eukaryotic)
•Conjugation (prokaryotic)
16