Download Solutions to Make for Cell Culture of Xenopus Oocytes

SOLUTIONS FOR MAKING SOLUTIONS!
Your experiments depend upon the quality of your chemicals and your ability to make up
the solution with exact concentrations. We will view a lab safety video.
What is a buffer?
Buffers negate acids or bases and keep the pH of the solution constant (however, buffers
can be overcome and pH changes take place if there is too much acid or base added –
exceeding the buffer capacity.
Make sure that you review what pH is….(test yourself: it refers to the concentration of
what? It is defined as…). We will use HEPES buffer.
Why do you need one?
When you break open cells, you break open lysosomes and they release acid. Acid will
denature (and inactivate) your enzymes. So buffers prevent a change in the cellular pH
when the acids are released during homogenization. Also, enzymes typically require a
specific pH value for optimal activity so buffers are needed to set that pH.
Choose a buffer:
Do you want to buffer a solution at cellular pH (perhaps 7.2)? or extracellular pH
(varies)?
Pick the buffer based on its pKa.
For a cellular pH solution (7.2), you would want a pKa of about 7.2. However, if you
expect the pH to drop during an experiment (acid is produced during the chemical
reaction?), then choose a buffer with a slightly lower pKa than 7.2.
The farther the pH from the pKa, the poorer the buffer buffers. A rule of thumb is that
buffers work well if you stay within 1 pH unit of the pKa.
Other factors to remember when choosing a buffer:
The buffer should have low binding of ions (if the buffer molecule binds calcium ions,
then the buffer may lower the concentration of ions way below what it should be).
The buffer should not change its pKa much with changing temperature. Often, buffers
are stored in the refrig or freezer; at lower temperatures, the pKa for some buffers really
really changes. This can cause problems if you make up the buffer at room temperature
and then cool or heat up (to body temp 37 C) the solution for the experiment. If you
make up Tris in a cold room at 4 C to a pH of 7.0, then use the Tris solution in a reaction
occurring at 37 C, the pH will have dropped 1.05 units to pH 5.95.
Some buffers are supplied as free acid/base (that is, the powered buffer can be a base;
will raise pH when you put the powder into solution- or an acid) or as a salt (chemical
companies add sodium or potassium to the base form). If you want a solution that
mimics the cytoplasm, which ion would you choose – Na or K?
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Is the buffer excluded by membranes? Have high solubility? Absorbs light between 240
and 700 nm (if you have an enzymatic assay that utilizes optical absorption, then this is a
problem).
Buffers are typically made up to 15 mM up to 50 mM (what is the need for buffering
capacity? If you need a lot, use 50 mM but most of the time, 15 mM is enough).
Tris is a poor buffer below pH 7.5, precipitates or binds many polyvalent ions like
calcium, and can often inhibit enzymes. Furthermore, Tris can pass through membranes.
Zwitterionic Good buffers are recommended (they cannot pass through membranes). If
you dilute Tris 10 fold, the pH of the tris solution will decrease about 0.1 pH- most
buffers however do not change their pH much with dilution.
See the list of Zwitterionic buffers in the table (note that some are not Zwitterionic).
What is the pKa of HEPES? What is the range of pH that will this buffer work?
IONIC COMPOSITION OF SOLUTIONS
Solutions typically have an ionic composition that mimics the tonicity (ionic strength) of
the cytoplasm, or provides for maximal activity of an enzyme. NaCl or KCl are used to
set the optimal ionic concentration- if the ionic composition is wrong, cells or organelles
like mitochondria will blow up. In addition, ions like Ca or Mg must also be present as
an enzyme requires it as a cofactor (remember, that amino acid charges are used in the
active site; sometimes the amino acid charge on the positive nitrogen is not powerful
enough, and the enzyme must capture an ion, Ca, Mg, Fe, to provide a stronger positive
charge; trace elements).
Review your text: Box 8A Osmosis; the special case of water diffusion.
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Review these terms- which one should your buffer be? Which one will cause the cell to
burst? To shrink? To remain the same size? Why? (HINT: where is the highest
concentration of water- inside the cell or out, and water moves from high to low
concentration).
Hypotonic:
Isotonic:
Hypertonic:
Osmolarity means….
STEPS FOR MAKING A SOLUTION
1. Write down all solutions that you will need and the final concentration needed.
2. How much solution do you need? 500 µL? 5 mL? 100 mL?
(note that 1 µL is 10-6 Liters; mL or ml is one thousandth of a liter or 1000 µL. A liter is
about one quart).
3. Do you want to make the straight solution or a concentrated solution? Typically, for
Ringer’s solutions, we make a 5X solution, store it in the freezer, and then thaw it and
dilute it to 1X for use that day. Other solutions are too valuable to make up 5X and are
made up 1X.
For example, if [NaCl] is to be used at 50 mM, we might make up a 10X solution at 500
mM.
Problem: make up a 5X stock solution of SDS, put in freezer, the SDS comes out of
solution; make sure that you really warm up the 5X stock solution and all the SDS goes
back into solution before you use it.
4. Check whether any chemical is hazardous. Labs should have the MSDS sheet for each
chemical (typically in a large binder) and you should read the label on the bottle.
Symbols on the bottle are for: health hazard, fire hazard, reactivity, etc. Check the Merck
Index if you are still not sure of possible dangers. You may need to wear gloves, eye
protection, a mask (power dust from SDS can irritate your nose) or make up the solution
in the fume hood. PMSF is a protease inhibitor and is poisonous, Acrylamide is used for
gels and is a carcinogen/neurotoxin, phenol burns skin, and ethidium bromide is a DNA
dye and mutagen.
5. What kind of water is to be used? Ultrapure reagent grade water is made with special
filters in the lab (filters require being changed every so often) and is required for cell
culture. Tap water is not to be used, and most labs have tap deionized water. Somewhere
in the building is a deionizer (series of ion exchange columns) that may be working fine
and purifying water, or the filters may be old, clogged or even breaking down releasing
the ion exchanger column material into the water. Some buildings may actually have a
distillation apparatus and this is very good water but not good enough for cell culture.
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EQUATIONS FOR CALCULATING LABORATORY SOLUTIONS
Different procedures are noted below:
1. Need Concentration or amount per volume.
Amount:1 mole of a substance is defined as the weight of the substance in grams numerically
equal to the molecular weight. That is, moles are a unit of weight; a balance is used to measure
moles. Atomic weight is weight of an atom, molecular weight is weight of all atoms (given in
Daltons).
Moles = g/molecular weight = a quantity
1 mole of glycine = 75 g
0.2 mole of valine = (molecular weight: 117 g) x 0.2 = 23.4 g
Note that mmoles = mg/molecular weight
1 mmole (millimole) is one thousandth of a mole
75 g of glycine is one mole or 1000 mmoles
0.2 moles of valine is 117 x 0.0002 = .0234 g or 23.4 mg
1 µmole (micromole) is 1 millionth of a mole
µmoles = µg/molecular weight
1 µmole of glycine = 75 x 10-6 g = 75 µg
There is no abbreviation for mole; the word must always be written in
its entirety.
Concentration or Amt per volume:
The abbreviation M is used only for molarity (note that it is underlined), never for moles.
1 M glycine solution = 1 mole glycine per liter = 75 g glycine per liter.
0.4 M valine solution = 0.4 moles valine per liter = 46.8 g valine per liter.
1 mole/liter = 1 mmole/ml = 1 µmole/µl = 1 M.
....
Notice that mM is not mmoles /ml but rather mmoles/l. The term M, even when preceded by an
m or µ is defined on the basis of one liter.
1 mM glycine solution = 0.075 g per liter = 75 mg per liter = 0.075 mg per ml = 75 µg per ml.
Do not confuse moles and molar (M). They are entirely different units; and only molar is
underlined. The former is as weight, the latter -concentration (weight per volume).
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2) How many grams of a chemical is needed for making up a solution? You want 500 ml of
a 0.5 M solution? Use the following equation:
(volume in liters) x (molecular wt, g/mole) x (concentration in molarity)
Note that the units cancel: this is the way that you can check to make sure that you have
calculated properly:
liter x (gram/mole) x (mole/liter) = grams
3) How do you dilute a stock solution? What is the volume of a 1.5 M stock solution needed to
make up 500 ml of a 300 mM solution?
Note that you have to put all numbers in same units (use liters not milliliters, etc). First, find the
dilution factor:
DILUTION FACTOR = (STOCK CONC.)/(FINAL DILUTE CONCEN.)= 5
TOTAL FINAL VOLUME OF THE DILUTE SOLUTION/(DILUTION FACTOR of 5)=
this is equal to the VOL. OF STOCK TO BE ADDED
So add 100 ml of 1.5 M stock to a graduated cylinder, then add 400 ml of distilled water to top
off to 500 ml total.
Note that how accurately you calculate, and how accurately you measure may mean the success
of your experiment. These factors are another source of error in experimenting. We will see
that use of pipettes is another source of variability and error.
REMEMBER TO USE GRADUATED CYLINDERS TO MEASURE OUT YOUR VOLUME;
REGULAR BEAKERS ARE NOT ACCURATE ENOUGH!!!
THE MOST ACCURATE WAY TO MEASURE A VOLUME IS BY WEIGHT: PUT ONE ML
OF WATER INTO A WEIGH BOAT, AND SEE WHAT IT WEIGHS! IT WILL BE ABOUT
1 GM – IF IT IS 1.0018 GRAMS THEN YOU ACTUALLY PIPETTED OUT 1.0018 ML OF
WATER. NOTE THAT WATER WILL EVAPORATE WHILE IT IS BEING WEIGHED!
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From “At the Bench” (by Kathy Barker)
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Sometimes molarity is not used, but a “percent solution” is:
Mixing Solutions:
If possible, use a stirring motor and stirring bars to mix solutions.
Put the container (weigh paper or boat) onto scales and tare it out (make the scale read
zero). Then add the power. Use a new spatula with each chemical; wash the spatulas
carefully between use so that you do not contaminate one chemical with another.
Store chemical in plastic container with lid, in fridge.
Know how to use the stirring motor, the pH meter and the scales.
First, you mix up all the power into a volume less than what you want; say 700 ml instead
of one liter. Then you titrate the pH to the proper value –adding either acid or base
depending upon the buffer used. Then when the pH is correct, top off the solution to the
final volume. Typically, you use a graduated cylinder or flask (not a beaker) with a
stirring bar rotating in the container.
More detail below.
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Mixing and the pH meter: From “At the Bench”
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If readings erratic, also try stirring solution while you are measuring pH and adding
acid/base, rinse the electrode to remove any salt encrustation (waving your hands over
salt encrustation can cause changes in the readings!!), and make sure that the water you
are using is not very cold or hot (is the hot plate heating up the solution?)!
If solutions become yellow, or moldy (see any precipitate when you swirl? Is it colored?
If the precipitate goes away with heating, then it probably is not mold. But, when in
doubt, throw it out!
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Pipetting:
The major skill of biochemistry, and sloppy pipetting can ruin your experiment.
Round, Rubber repipette bulbs:
First, you want to push air out of the bulb by squeezing A for aspirate (at the top), then
draw liquid into the bulb by pushing S for suction, and to let the fluid flow out, push E
for exhaust or expel. Use with glass pipette: for volumes >1 ml (e.g.:~5, 10 ml).
Micro- Pipettors (Rainin Pipetmen, etc):
Typically come in these three sizes: 1 to 10 microliters, 10 to 100 microliters, and 100 to
1000 microliters (or one ml). Place tip on the end (use a new plastic tip for every new
solution so there is no contamination), push button on top down with your thumb, then
place tip into solution (meniscus about where the liquid will go up to in the plastic tip),
then SLOWLY let the button on top up. Repeat (as the first fill is thought to wet down th
sides of the plastic tip and the second filling is more accurate). Fill the tip too fast, and
you will not pipette accurately (you can place the solution on the scales to see how
accurate you can pipette 5 µL).
Then place the tip in the new solution and expel, draw up solution and expel again to
wash out the tip.
Solutions to Make for Cell Culture of Xenopus Oocytes:
Go through steps; how many ml do you need for an experiment? You will put the cells
that I give you into Petri dishes (with lids- takes about 15 ml per dish), and keep the cells
alive for as long as you can at room temp. Human cells require higher temp (37 C) and
special gas over the cells. Solutions to make up for oocyte culture:
1. Making up the solution – called O-R2- that we keep Xenopus oocytes in: an
inexpensive cell culture system. O-R2: so the cells do not swell up or shrink (correct
tonicity).
Make up 500 ml of a 5X stock solution that will be stored in the frig or freezer. Then
when you want to use a solution, you take out the 5X stock, warm it up to room
temperature and then dilute five times.
The pH will be adjusted when you make the 5X stock solution but the pH should be
rechecked after it is thawed and diluted (it should be 7.9). Remember cold solutions
might give you a bad pH reading; so make sure the solution is at room temperature.
a. Make the 5X stock solution
How to make 0.5 L of the 5X stock solution:
Ion
1X final conc.
5X; g in 0.5 L
MW
NaCl
83 mM
_______ g
58.44
CaCl2
0.5 mM
_______ g
147.02
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MgCl2
1 mM
________ g
203.3
Hepes
10 mM
________ g
238.3
Buffer (free acid not sodium salt; Na HEPES has a MW of 260.3)
SHOW YOUR ANSWER AND CALCULATIONS IN YOUR LAB NOTEBOOK.
We typically leave K out of the solution because the cells respond faster to
hormone.
Add powder into an appropriate half liter container, add water to only about 250
ml. Before topping off the solution to 0.5 L, titrate to pH 7.9. Do you have to use a base
or an acid? HEPES is the buffer; look up its pKa and note it in your lab notebook.
b. To Make Up the Working solution (1X):
If the stock has been kept in the refrigerator-make sure to warm it up to room
temperature. Take out 100 ml and dilute 5 X to what final volume?
You might not top it off, but check heck the pH after it is at room temperature (it
should be 7.9) and adjust if necessary- then top it off to the correct final volume.
2. Insulin: We will make up a solution of insulin and then add the hormone to the
Xenopus oocytes and watch for induction of meiotic cell division.
First, know a little bit about handling proteins. You have to worry about trace amounts of
degradative enzymes (a protease) destroying your protein. So, always keep your protein
solutions on ice (typically, use a large bucket with ice in it)- this slows down all chemical
reactions including that of the degradative proteases. Also, you may have to add protease
inhibitors (PMSF, aprotinin, leupeptin, etc).
Keep the protein solution cold even while performing various steps such as
centrifugation; typically the centrifuge is kept in a large cold box (like those in stores to
keep pop or beer), or in a special cold room (like a meat locker).
Proteins may be easily denatured by heat or by repeated freezing and then thawing.
Read below: HANDLING PROTEINS (from Boehringer Mannheim Biochemicals):
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Procedure for making up insulin:
NOTE WHEN MAKING UP ANY SOLUTION IN THE LAB, at least put THE DATE,
YOUR INITIALS AND THE FORMULA/what it is on the container. Use a nonearable
pen and then put clear tape over it. Then record all OF YOUR CALCULATIONS IN
LAB NOTEBOOK SO THAT WE CAN FIND OUT WHAT YOU DID A MONTH
FROM NOW.
Make up a solution of 2 mM HCl to dissolve insulin in. How would you do this from the
solution that I give you?
What is the concentration of concentrated HCl? How many fold would you have to
dilute it to get to 2 mM HCl?
Add 9 mg of insulin to 10 ml of 2 mM HCl. Weight as accurately as you can. Measure
the 10 ml as accurately as you can (use what pipette?)-- Can you weigh out the 10 mlwould this be more accurate?
Note in your lab notebook how you measure out 9 mg on the scales provided.
Make sure that the insulin powder goes into solution; if not, add a little bit more of 10
mM (not 2 mM) HCl.
If insulin’s molecular weight is 6000 g, what concentration of insulin did we just make?
SHOW Calculations:
Next week, we will check your accuracy by measuring insulin concentration with a
spectrophotometer.
3. Antibiotic: Since the cells are in nice medium, other things can also grow rapidly.
So, add Gentamycin: antibiotic; make a stock of 5-10 mg/ml for a final concentration of
50 µg/ml. Aliquot the stock in 1 ml V vials (or Eppendorf tubes), store at -20 C in a
nondefrosting freezer (defrost refrigerators, also called frost free refrig, actually heat up
at night to get rid of frost, and this constant freezing/thawing to warm temps will kill the
antibiotic and enzymes). Dilute stock 100 fold.
4. Carbon Source The cells need a carbon source so that they can make ATP. So, add
a little pyruvate (or pyruvic acid; where does it enter respiration?).
Sample Questions to answer: (others to follow….)
Why is Tris hard to pH?
With the HEPES buffer, did you add base or acid to make the O-R2?
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