Lab Techniques &

Storage of Extracted NA
BIO 206
Laboratory Practices
Pipettors:
◦ Always check for correct volume
◦ Place at max volume after use

Centrifugation:
◦ Microfuge tubes must be of equal weight
◦ Well-balanced
Laboratory Practices
UV Spectrophotometry/ Nanodrop:
◦ Use Kimwipes w/ 70% ethanol before and after
◦ Do not touch well with tip

Gel Electrophoresis:
◦ Use Kimwipes w/ 70% ethanol before and after
◦ Place wells near the (-)
◦ Be careful not to pierce beyond the well
Safety considerations
Chloroform is toxic and a suspected human carcinogen.
◦ harmful if inhaled, ingested or exposed to the eyes or skin.

Sodium Dodecyl Sulfate (SDS) is harmful if inhaled or ingested. It potentially causes skin and eye
burns.
◦ Wear a dust mask or respirator;
◦ Prepare in a fume hood
Storage
DNA RNA

-DNA is one of the most stable molecules -less stable than DNA
known Degradation during storage = unusable for
-few applications: downstream assays
◦ newborn screening for a genetic disease ◦ reverse transcription
◦ in vitro translation
◦ analyze forensic evidence
◦ differential display
◦ study a gene
◦ expression array and expression-chip analysis

-pure samples are always better esp. for storage

Threats to stored DNA and RNA:

nucleases, oxidation due to trace amounts of metals
Treating Solutions with DEPC
[WARNING: DEPC is a suspected carcinogen.
◦ Take appropriate precautions when handling; e.g., always wear gloves and handle under an approved
fume hood]

DEPC reacts with histidine residues of proteins and will inactivate Rnases

Protocol:
Add DEPC to solutions at a concentration of 0.05 - 0.1%
stir or shake into solution, incubate for several hours
autoclave at least 45 minutes, or until DEPC scent is gone.
Storage temperatures
for DNA preservation:
◦ Room temperature on a ‘dry’ solid matrix
◦ –20°C
◦ –80°C
◦ –196°C (storage in liquid nitrogen)

for RNA preservation:

◦ –20°C
◦ –80°C
◦ RT
The glassy state
For temperatures: RT and -196 C
◦ Said to last for decades

-196 C:
◦ Maintained in a vitreous state

If moisture is added to the ‘dry state’ or the temperature is raised:

◦ movement and reactivity of protons is re-established
◦ damage to the DNA can occur
Vitrification
- increasing the solution concentration
- lowering temperature

store dried DNA samples at: low relative humidity

two methods:
◦ freeze drying
◦ spray drying
Freeze drying
DNA (1 mg /mL) was dissolved in Tris-edetate buffer (TE buffer)
dispersed in a 1% w/v solution of PLGA in dichloromethane.
added to 1 L of 1% polyvinyl alcohol (PVA) and emulsified using a Silverson L4R Mixer (Silverson,
Bucks, UK).
evaporation by stirring at a controlled temperature for 12 h,
microparticles were recovered by centrifugation
washed three times in de-ionized water.
lyophilized by immersion in a Hetofrig cooling bath (Heto) for 15 min
transferred to a Hetosicc CD52 freeze-drier (Heto) and left overnight
Spray drying
Same emulsification technique with freeze drying except for last two steps

Dispersions were spray dried in a Buchi mini Spray Dryer Model 191 (Buchi Laboritorium-
Technik, AG, Flawil, Switzerland) using an inlet temperature of 78/79C

aspirator rate of 75%, a pump rate of 10% and an airflow rate of 600 NL/h
Room Temp
dried DNA on FTA Cards (Whatman)
The DNA immobilized on FDA cards is amenable to PCR after 17 years storage

RT for RNA:
Lasts up to a year
Reagents:
◦ RNase-free H2O (with 0.1 mM EDTA)
◦ TE buffer (10 mM Tris, 1mM EDTA)
DNA: -20 to -80 C
Several months (-20C) to several years (-80C)

Usually done in laboratory settings

DNA in the aqueous phase is stored under slightly basic conditions in

◦ Tris EDTA buffers (pH 8.0 in -20 C)
◦ or as a precipitate under ethanol (at -80° C)

NOTE: to avoid repeated freeze-thaw cycles, often used samples are placed on -4C.
RNA: -20 to -80 C
Stored as ethanol precipitates
must be pelleted and dissolved in an aqueous buffer before pipetting

Reagents:
◦ Precipitated in ethanol (~2 years)
◦ solubilized in formamide (-20°C, no degradation for a year)
Caution on Freeze-Thaw cycles
Limit freeze-thaw cycles to less than 10

Damage on the NA
◦ pH
◦ Physical shearing by ice crystals
◦ Oxidative damage catalyzed by metal ions

Bad sample:
◦ incompletely purified state with excessive metal ions
◦ unbuffered water