Buffers

and
Reagents

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 Group No: 3
• Laiba Ashfaq
• Anam Asif
• Saadia Usman
• Alina Maheen
• Farwa Rasool
• Summaya Bibi
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DEFINITION:
Buffers are compounds or mixtures of
compounds that by their presence in the solution
resist changes in the pH upon addition of small
quantities of acid or alkali.

WHY DO WE NEED BUFFERS?

•  For the preservation of a solution upon
storage
• To avoid the effects of external influences that
cause a change in the pH
• Helpful in the pharmaceutical sector
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PREPRATION OF BUFFERS:
• The buffers can be prepared by mixing a weak (poorly
dissociating) acid with its strong salt
• For example: by mixing acetic acid and sodium
acetate.
• One part of the buffer can neutralize protons and
other part hydroxyl ion. Therefore, these solutions can
effectively slow down pH change

BUFFER ACTION:
The resistance of a buffer solution to a change in pH is
known as buffer action.
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 MAKING REAGENTS AND BUFFERS

SAFTEY:
before making any buffer or reagent, we must check the hazards
associated with handling of material
• Gloves: gloves should be used for handling reagents
• Latex or PVC gloves provide good protection against most powdered
reagents
• Rubber or neoprene gloves are chemical resistant and are used for
handling organic solvents
• Eye protection : plastic glasses or goggles should be used for protection
against splatters, aerosols, combustible or breaking glass.
• Hood: always make volatile substances in chemical hood.
• Mask: simple dust-mist or surgical mask is enough for protection
against powders. For volatile substances use respirator masks
• Labels: label all the bottles with the hazard associated to them

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 Which water to use?

• Water is an important
component of solutions
• Water quality, composition
and pH drastically effects
experiments
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 Classifications of water quality
• Tap water:
*tap water quality depends upon geographical location,
pipes and rainfall
*some minerals can inhibit enzymes, so it is only used for
washing glassware and other laboratory equipments.
• Laboratory grade water:
* it is purified by reverse osmosis or distillation.
*it is used for making buffers
• Reagent grade water:
*laboratory grade water is further purified through
distillation or deionization.
* It is used for cell culture, biochemical reagents and buffers
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Ultrapure reagent grade water:
* Water treated by ultrafiltration is endo-toxin free.
*This may be required for certain cell cultures.
*it is filtered through a combination of reverse osmosis,
ultrafiltration, deionization, adsorption and ultravoilet
oxidation.
* It can also be purchased from scientific and hospital
distributors.

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 Weighing and mixing
Weighing out ingredients:
• It is the most important part of the experiment.
• Gather everything that is required before starting
experiment, to avoid leaving your powder uncovered
Required apparatus:
• weighing paper
• Weighing boats
• Spatulas
• A clean graduated cylinder
• Beaker or Erlenmeyer flask
• Magnetic stir plate and stir bar
• Kimwipes
• Distilled water
• Place to dispose off spatulas, stir bars 9
 Sterilizing solutions
Autoclave:
• Most buffers
• Undefined bacterial and yeast media.
Do not autoclave:
• Buffers with detergent such as 10% SDS, because they will boil over.
• Organic solvents including phenol
• Heat labile compounds such as serums and vitamins, antibiotics and
proteins
• Mammalian, plant and insect media
• HEPES containing solutions
• DTT or BME containing solutions
Filter sterilization:
• if a liquid is heat labile or volatile or less than 20ml in volume it should be
sterilized through filtration.

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 Preparation of buffer stocks
TBE buffer: It is used for
• DNA and RNA polyacrylamide gel electrophoresis
• Non denaturing and denaturing acrylamide gels
• DNA automated sequencing gels
• STR analysis and RSCA
• DNA agarose gel electrophoresis
TAE buffer: it is used for
• DNA agarose gel electrophoresis
• Non denaturing RNA agarose gel electrophoresis

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 TAE buffer vs. TBE buffer
TAE Buffer
• It consists of tris base, acetic acid
and EDTA.
• It is commonly prepared as 50X
concentrated stock.
• It has better conductivity than TBE,
so DNA fragments will migrate faster
in it.
• Provides better resolution of smaller
DNA fragments.
• It is preferred when using DNA for
any downstream applications like
PCR and clonal ligation.
TBE Buffer
• It consists of tris base, boric acid and
EDTA.
• It is commonly prepared as 10X
concentrated stock.
• It has better buffering capacity than
TAE buffer, used for extended long or
repeated runs.
• Provides better resolution of larger
DNA fragments.
• It costs more than TAE.
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ANY QUESTION?

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