Laboratory 4a
Making Solutions for DNA Isolation
Background
For genetic engineering, PCR, or other work with DNA, a pure DNA sample is required. DNA
must be purified from cells, removing all other cellular constituents and contaminant molecules.
Many purification protocols include a step to remove protein contaminants using a salt solution.
One of the final steps in DNA isolation is to precipitate DNA, or take the DNA out of solution.
In most cases, DNA precipitation is done using alcohol. In several upcoming laboratory activities,
DNA strands will be precipitated from solution. In Laboratory 4b, genomic salmon testes DNA
will be precipitated onto a glass rod. To increase the number of DNA strands that will spool
around the glass rod, 5 M sodium chloride (NaCl) is added to the solution prior to alcohol
precipitation. The Na+ ions in a NaCl solution bind to the DNA, decreasing its negative charge,
allowing DNA molecules to come closer together and spool more easily.
Isolated DNA can be stored for long periods in sterile TE buffer (containing TRIS and EDTA).
The TE buffer contains TRIS to maintain the pH of the DNA sample and EDTA to denature any
DNases, which might contaminate the sample. In this activity, you will prepare 5 M NaCl solution
and TE buffer.
Purpose
To make 10 mL of 5 M NaCl solution.
To make 100 mL of TE buffer (10 mM TRIS, 1 mM EDTA, pH 8.0)
Materials
Balance, analytical
Balance, tabletop milligram
Weigh paper, 7.6×7.6 cm
Weigh boat, 3.5”×3.5”
Lab scoops
Sodium chloride
Tubes, 15 mL, conical
Tube racks for 15-mL tubes
TRIS
EDTA, disodium salt
Media bottle and cap, 125 mL
pH paper, wide/narrow-range
Hydrochloric acid
Sodium hydroxide
Glass rods
Procedure
Part I: Preparation of 5 M of NaCl
1. Determine the mass of NaCl to be measured. Remember, you want enough NaCl to give a
concentration of 5 M, but you only want to make 10 mL of this solution. In your notebook
show the calculations and draw a diagram of how the solution will be prepared.
2. Weigh the amount of NaCl needed and place it in a 15-mL conical tube. Slowly add dH2O,
while stirring, until a final volume of 10 mL is reached.
3. Cap the tube and invert it for several minutes until the salt dissolves completely into solution.
Check to make sure that the volume is still 10 mL. If it is not, add a tiny amount of dH2O
until the final volume is 10 mL. Label the tube with the sample name, concentration, date,
and technician’s initials. Store at 4°C until ready to use.
Part II: Preparation of TE Buffer
Note: The calculations for each solute (TRIS and EDTA) are done separately based on a final
volume of 100 mL.
1. Determine the mass of TRIS to be measured (from the bottles in the chemical storeroom)
to give the correct concentration and volume in the final TE buffer. Show the calculations in
your notebook.
2. Determine the mass of EDTA to be measured (from the bottles in the chemical storeroom) to
give the correct concentration and volume in the final TE buffer. In your notebook show the
calculations and draw a diagram of how the TE buffer solution will be prepared.
DNA Isolation and Analysis
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