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 2l 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
