MATERIALS

EXPERIMENT 1A:

PROPERUSEOFMICROPIPETTES,MICROCENTRIFUGEANDOTHERBACIS
MOLECULAR WORK EQUIPMENTS

 A set of three pipettes per group (2 - 20μl, 50-200μl, and 200 – 1,000μl)
 Three boxes of corresponding pipette tips per group (white, yellow & blue)
 Microcentrifuge tubes per group
 Beaker per group for pipette tip waste
 Soil suspension (for centrifuge & pipette practice) Latex gloves
 Microcentrifuge 6X gel loading dye
 Ficoll (type 400)
 1% bromophenol blue (BPB) 100% glycerol
 Tris base
 Acetic acid glacial
 0.5 M Ethylenediaminetetraacetic acid (EDTA) solution.
 Tris base
 Boric acid
 0.25% bromophenol blue
 0.25% xylene cyanol
 FF 15% Ficoll Type 400
 120 mM EDTA

EXPERIMENT 1B: PROPERUSEOFMICROPIPETTES,MICROCENTRIFUGE AND

OTHERBACIS MOLECULAR WORK EQUIPMENTS.

 Overnight bacterial culture

 1X Tris-EDTA (TE) Buffer:
 10 mM Tris-Cl pH 7.4 1 mM
 Ethylenediaminetetraacetic acid (EDTA).
 1-liter ddH20
 10% sodium dodecyl sulfate (SDS)
 20 mg/ml proteinase K (stored in small single-use aliquots at -20◦C)
 5 M NaCl
 CTAB/NaCl solution (10% CTAB in 0.7M NaCl,).
 Chloroform/isoamyl alcohol (24:1)
 Phenol/chloroform/isoamyl alcohol (25:24:1)
 Isopropanol
 70% ethanol
EXPERIMENT 1C
AGAROSE GEL ELECTROPHORESIS (AGE) ANALYSIS

 TAE Buffer (50X)

 TBE Buffer (10X)
 Loading Dye Buffer (6X)
 Agarose powder
 Standard DNA Ladder marker
 Sterile ddH2O
 10 mg/ml ethidium bromide (EtBr). (stock solution)
 Agarose Gel Electrophoresis System
 DNA samples (bacterial plasmid & chromosomal DNA)

PROCEDURE

EXPERIMENT 1A:
PROPERUSEOFMICROPIPETTES,MICROCENTRIFUGEANDOTHER
BACIS MOLECULAR WORKEQUIPMENTS

a) Pipetting Technique:
1. The most appropriate volume pipette and corresponding tip is choosen.
2. The volume on the pipette is adjusted by turning the knob to the desired volume.
3. The pipette is depressed until the resistance is felt. The pipette tip is inserted into liquid
and released.
4. The pipette is depressed again carefully to discharge the liquid to avoid the contamination
the tip during transfer.
5. The pipette tip is released into desired tube by pressing white button. The volume capacity
specified on the pipette is never go beyond
6. The liquid solution of 1000µl, 500µl and 10µl is pipetted. The right micropipettes is
used, each type of micropipette is adjusted to the right aspiration volume and the right
kind of micropipette tips is used.
7. Using the micropipette system, serial dilution of 10X and 100X is performed.
8. 15 l of solution is loaded into an agarose gel provided. The right proportion of 6X
GLD (Gel Loading Dye) to your solution is added to the solution. The solution for
loading contained X GLD.
 b) Centrifuging Technique:
1. The centrifuge tubes are placed directly across from each other in the centrifuge.
2. The centrifuge is critically balanced. The tubes have similar volume of sample and an
even number of tubes. To balance the centrifuge, an extra tube with water may add.
3. Coordinate with another group to combine tubes in order to balance and would save
time.
4. The spray guard is replaced, the lid is closed and desired rpms and time is set.

c) Protocol for separating clear fluid from a cloudy mixture (Pipette & Centrifuge Practice)
1. The soil suspension mixtures are swirled and 500μl from each mixture is aliquoted into
labelled centrifuge tubes.
2. The mixture is centrifuged at 12,000 rpms for 1 minute (BALANCE!).
3. The supernatant is off as much pipetted carefully into a fresh labelled tube.
4. The steps 2-3 are repeated with the supernatant obtained until a clear liquid is formed.
The steps are repeated for all tubes.

EXPERIMENT 1B: PROPERUSEOFMICROPIPETTES,MICROCENTRIFUGE AND

OTHERBACIS MOLECULAR WORK EQUIPMENTS.

1. A 5-mL liquid culture is inoculated with the bacterial strain of interest. That strain is grow
in appropriate conditions.

2. 1.5mL of the culture is spin in a microcentrifuge for 2 minutes or until a compact pellet
forms. The supernatant is discarded.

3. Pellet in 567 µL TE buffer is resuspended by repeated pipetting. 30 µL of 10% SDS and 3 µL of

20 mg mL-1 proteinase K is added to give a final concentration of 100 µg mL-1 proteinase K in
0.5% SDS. It is mixed thoroughly and incubated for 1 hour at 37°C.

4. 100 µL of 5M NaCl is added and mix thoroughly.

5. 80 µL of CTAB/NaCl solution is added. It is mixed thoroughly and incubated for 10

minutes at 65°C.

6. An approximately equal volume (0.7 to 0.8 mL) of chloroform/isoamyl alcohol is added,

mixed thoroughly and spinned 4 to 5 minutes in a microcentrifuge.

7. Aqueous, viscous supernatant is removed to a fresh microcentrifuge tube, the interface is

left behind. An equal volume of phenol/chloroform/isoamyl alcohol is added, extracted
thoroughly and spinned in a microcentrifuge for 5 minutes.
8. The supernatant is transferred to a fresh tube. 0.6 vol isopropanol is added to precipitate
the nucleic acids. The tube is shaken back and forth until a stringy white DNA precipitate is
clearly visible. At that point, the pellet is possible to transfer to a tube containing 70% ethanol
by hooking it onto the end of a micropipette that has been heat-sealed and bent in a Bunsen
flame. Alternatively, the precipitate is pelleted by spinning briefly at room temperature.

9. The DNA is washed with 70% ethanol to remove the residual CTAB and spun again 5
minutes at room temperature to repellet it. The supernatant is carefully removed and briefly
air dried the pellet at room temperature. The pellet is redissolve in 100 µL TE buffer.

EXPERIMENT 1C

AGAROSE GEL ELECTROPHORESIS (AGE) ANALYSIS

a) Preparing mini agarose-gels

1. Agarose in buffer is prepared ahead of time and the solution is stored in small Erlen
meyer flasks for later use. The agarose gel solution melted in a microwave oven prior
to pour. The solidified agarose gel can be stored at room temperature for several
weeks.
2. The appropriate size of mini-gel rig/comb combinant is choosed based upon the
number of samples to be run. 30mL Owl Scientific rigs (Model B1A) have combs to
accommodate either 6 or 10 samples. ‘Double load’ also can be placed two combs in a
single gel.
3. The gel mold is turned so that the gaskets are against the inner walls of electrode
chamber. The comb is positioned in the mold. (depth preset).
4. Heated the flask with agarose gel solution using microwave, occasionally checked to
see whether all the agarose has melted. The agarose is melted in a beaker of water on
a hotplate if the microwave is not available.
5. The appropriate volume of hot agarose is measured into agraduated cylinder. The
cylinder can cooled by running cold tap water along the outside of the flask. The
agarose is poured into the mould when only warm to the touch. The gel rig is placed
in a refrigerator prior to pour the agarose to speed up the process of solidification.

b) Preparing samples and use of mini-gels

1. The comb is gently removed by pulling evenly upward.
 2. The gel mold is positioned in the electrode chamber and covered with gel running
buffer.
3. The samples are loaded into the lanes below the meniscus of the buffer solution since the
agarose gel is submerged beneath electrophoresis buffer.
4. Use a piece of Parafilm to mix the dye with the sample. The number of samples is
written on the Parafilm using a permanent marker to be analysed. 10 µl of dye is
spotted near each of the number. An extra spot for the molecular weight standard that
is used is added next to the samples.
5. 4 µl of sample is pipetted and added to the dye spot of the correct number. The tip is
holded and ejected. The tip is placed on another pipette set to 14 µl and the entire
sample/dye mixture is draw. The mixture is pipetted to the appropriate mini-gellane
so that photographed lane number one is to the top left. The process is continued until all
the samples and the molecular weight standard DNA marker is loaded.
6. The cover is placed to the electrode box and the wires are plugged to the power bank.
The samples are migrated migrate towards the red or anode pole. Power pack is turned
on and the low milli-ampherage setting is used and electrophoresis at 75 milliampheres
(mA) is started. The samples are not allowed to move too fast or the apparatus to
become too hot. Run times vary slightly. The Owl rig system took half to an hour
depending on the experiment. Electrophoresis is stopped before the bromophenol blue
dye exits the gel (about 2 cm from the end of the gel).

c) Staining and Photographing Mini-gels

1. The gel mold is gently removed from the electrode chamber after switching off the power
and the gel is slipped into the plastic staining box containing EtBr solution.

2. The EtBr stock solution is kept at 4 C in a dark bottle as a 10 mg/ml solution. 20 µl of EtBr to
40 ml of 1X TBE buffer is added when used. Stained for 15 minutes.

3. UV light is used to view EtBr-stained DNA. Protective eye goggles/glasses are wore when
the UV source in a transilluminator is switch on.

4. To photograph mini-gel, the transilluminator is turned to the highest setting and the
agarose gel photo is documented using digital camera.