Lab Protocols

Preparation of solutions
1. LB broth medium:
Weigh exactly:
+ Peptone/tryptone : 1g
+ Yeast extract : 0.5 g
+ NaCl : 1g
Dissolve all components in 80 ml H2O, then fill up to 100 ml. Autoclave the
medium at 121oC for 30 min.

2. Lysis buffer:
+ 400 mM Tris-HCl, pH 8.0
+ 60 mM EDTA, pH 8.0
+ 150 mM NaCl
+ 1% SDS
3. Other solutions: 5 M CH3COOK, pH 4.8; 100% Isopropanol; 70% Ethanol
4. PCR reagents:
+ 5 mM dNTPs
+ 5 M Primers
+ 10X Buffer
+ Taq DNA polymerase
5. Electrophoresis buffer: 1X TAE
+ Tris-base : 4.84 g
+ Glacial Acetic Acid : 1.09 g
+ EDTA : 0.29 g
Dissolve and fill up 1 L by H2O

6. Gram stain solutions

Primary stain: crystal violet
Mordant: iodine
Secondary stain: safranin
Decolorization solution: ethanol 96% (with iodine) or mixture of ethanol and
acetone
Practice 1: Observation of bacteria by microscope
Gram stain:
- “Heat-fix” the slide with the specimen by passing it over a heat source, such
as a flame, several times using the forceps. The slide should be passed very
quickly through the flame and not be heated excessively. Place slide on the
staining tray.
- Flood the fixed smear with crystal violet solution (#1) and allow to remain
for 1 minute.
- Rinse off the crystal violet with distilled or tap water.
- Flood the slide with iodine solution (#2). Allow to remain for one minute.
- Rinse off the iodine solution with distilled or tap water.
- Flood the slide with decolorizer (#3) for one to 35 seconds.
- Rinse off the decolorizer with distilled or tap water.
- Flood the slide with safranin (#4). Allow to remain for 1 minute.
- Rinse off the safranin with distilled or tap water.
- Dry the slide on bibulous paper or absorbent paper and place in an upright
position.
- Microscopically examine the slide for bacterial organisms under a 100X
objective. Observe several fields on the slide for bacterial organisms.
Describe the gram reaction of any organisms seen. Gram-positive bacteria
stain deep violet to blue and gram-negative bacteria stain pink to red.

Practice 2: Culture of bacteria:

- Prepare sterile vials in clean bench
- Add 2 ml of LB medium to each sterile vial
- Pick one colony from plate and transfer into each vial
- Incubate vials in incubator at 37oC with shaking 150 rpm overnight

Practice 3: DNA isolation from bacteria:

- Harvest the E. coli cell pellets from overnight culture by centrifugation at
5000 rpm for 5 min
- Resuspend the pellet in 500 ul lysis buffer. The tube is then left at RT for 10
min.
- Add 150 ul CH3COOK, pH 4.8 and mix well by inverting the tube up and
down
- Spin the tube at 10000 rpm for 1 min.
- Collect and transfer the suspernant to another 1.5-ml Eppendorf tube
- Add an equal volume of isopropyl alcohol and mix well by inverting the tube
briefly.
- Centrifuge at 10000 rpm for 15 min at 4 degree Celsius
- Collect and wash the DNA pellet with 300 ul of 70% ethanol, followed by
centrifuging at 10000 rpm for 5 min
- After washing, dry the DNA pellet by air and dissolve it in 50 ul of DI H2O
- Check the DNA extract by agarose gel electrophoresis
Practice 4: DNA quantitation by spectrophotometry:
- Turn on the NanoDrop system
- Blank the measurement by using DI.H2O
- Load 2l of DNA sample and measure
- Calculate the DNA amount

Practice 5: Agarose gel electrophoresis

1. Preparation of agarose gel
 Weight 0.8 g agarose into a 250 ml Duran bottle
 Fill up 100 ml by 1X TAE buffer
 Boil until agarose is dissolved completely
 Let cool down to 40-50oC
 Add DNA staining solution (1:20000X)
 Pour the solution into a prepared electrophoresis tray (comb inserted)
2. Electrophoresis
 Insert the agarose gel into the electrophoresis tray and fill it up with 1X
TAE buffer
 Withdraw the comb from agarose gel
 Load samples into spective wells
 Apply the current with 100 V constant until the dye front reach the end of
agarose gel
3. Gel documentation
 After electrophoresis, take the gel out of electrophoresis system and
submerge it in the ethidium bromide solution for staining 5 min
 Wash the gel with H2O by transferring the gel to H2O container
 View electrophoresis pattern by Geldoc system

Practice 6: PCR reaction:

PCR components:
1. 5 mM dNTPs : 1 l
2. 10x Buffer : 2.5 l
3. Primers : 2 l
4. DNA template : 5 l
5. MgCl2 : 2.5 l
6. Taq DNA pol : 0.2 l
7. H2O : 14.3 l
Total : 25 l

PCR cycling:
1. 94oC : 5 min
2. 94 oC : 45 sec
3. 56 oC : 60 sec
4. 72 C
o
: 90 sec, repeat from step 2 for 35 times
5. 72 oC : 7 min
Check PCR products by agarose gel electrophoresis