Professional Documents
Culture Documents
1
Solution problems Using stocks to make dilutions
Suppose that you’ve prepared these three stock solutions:
Sodium acetate has a FW of 82.03
1 M NaOH (FW = 40.0)
3. How many grams of sodium acetate would you weigh out 1 M NaCl (FW = 58.4)
if you wanted to make 100 ml of a 1 M solution? 10% (w/v) SDS sodium dodecyl (or lauryl) sulfate
4. What would be the molarity of the same solution if you Using these stocks, how (exactly) would you prepare these?
prepared it in 200 ml instead of 100 ml? 50 ml of a solution that is 154 mM NaCl?
10 ml of a solution that is 0.2 M NaOH, 1% SDS
Exactly what steps would you take to make this
solution correctly?
…then these all mean the same thing: 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
What are pH buffers? Example of acid/base pair
They’re made up of conjugate base and acid forms.
H+
pKa = 4.75 (acetate)
H+
Acid form Base form
http://course1.winona.edu/ sberg/ChemStructures/ Dissocia.gif
H+ H+
H2O
H+ H3O+
http://olw. mit.edu/media/glossary/tris.jpg
Read section 3.7 carefully
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
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?
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)
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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.
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
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
50% dissociated
at pH 9.47
(possibly an
50% dissociated
at pH 2.98 (possibly
DNA
empirical finding) an empirical finding)
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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-carrying cell Plasmid-free cell stops Plasmid-carrying cell Plasmid-free cell also
survives and propagates growing survives and propagates survives and propagates
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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
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
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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
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.
H3O+
0 _ 0 _ + _
_ + 0 + 0 +
_ 0 0
+
_ +
_ +
_
_ _ 0
0 _ + + _ + + _ +
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… 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…
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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.
plasmid
1% SDS (sodium dodecylsulfate)
0.2 N NaOH
ssDNA of
genome 5 M KOAc (pH = 4.75)
plasmid
Separate by centrifugation
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
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
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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
5’-...GGAGATCAGACTACGATCAGACAGCT...-3’
3’-...CCTCTAGTCTGATGCTAGTCTGTCGA...-5’
p. 133
Fig 4.7
15
5’ phosphate and 3’ hydroxyl are
required for T4 DNA ligase Introduction of DNA into cells
•Transformation (prokaryotic)
– “competent” cells
– CaCl2 vs. electroporation methods
(removes phosphate group)
•Transfection (eukaryotic)
•Conjugation (prokaryotic)
Fig 4.6
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