Tuesday, October 10, 2006



A list of sites with valuable protocol sheets:
Do you have a Protocol to share?
Send us the URL and we will post it for you.
2-D Gel Electrophoresis
Improved 2-D PAGE making process as well as post-separation analysis techniques.
Maintained at The Electrophoresis Laboratory at Geneva University Hospital.

Applied Molecular Biology Beginning Laboratory Manual
Maintained by The University of Maryland, Baltimore County

Arabidopsis Guide
Extensive guide to all that is Arabidopsis from Stanford University Genomic Resources.

Cell Biology Laboratory Manual
William H. Heidcamp Laboratory at Gustavus Adolphus College.

CGH Protocols
Comparative Genomic Hybridization (CGH), a molecular cytogenetic method of screening a tumor for genetic changes.
Published by Institut für Pathologie, "Rudolf-Virchow-Haus", University Hospital Charité.

Comprehensive Protocol Collection
A comprehensive collection of molecular biology and Nematode-related protocols at Dartmouth College.

Donis-Keller Lab, Lab Manual
Maintained at Washington University in St. Louis.

Field and Laboratory Protocols
From the Laboratory for Microbiological Oceanography at Hawaii.

Frontiers In BioScience Protocols Page
DNA and protein protocols.

General DNA, RNA and Protein Methods
From the Melbourne Signal Transduction Group.

Gerard R. Lazo Laboratory Protocols
Western Regional Research Laboratory at the USDA ARS.

Herskowitz Lab
Yeast Genetic Protocols.

Histology Lab Protocols
Over 100 histology protocols from the Nottingham Laboratories.

Long PCR Reagents and Guidelines
General Guidelines for Long PCR Conditions and Enzyme Mixtures
From the George M. Church Laboratory and the Lipper Center.

MBS - Molecular Biology Shortcuts

Melbourne Signal Transduction Group

Microscopy Techniques
From the Melbourne Signal Transduction Group.

Molecular Biology Protocols
Protocols for DNA, RNA and protein from the Northwest Fisheries Science Center WWW Server.

PCR and multiplex PCR guide
An extensive discussion on PCR techniques and variables.
Designed by Octavian Henegariu, Indiana University.

Production Sequencing Protocols
Used at the Stanford DNA Sequence & Technology Center.
Protein Mass Spectrometry Protocols

Protocols for recombinant DNA isolation, cloning, and sequencing.
"A compilation of many of the everyday methods used in the average molecular biology laboratory with emphasis on the techniques for large scale DNA sequencing protocols and DNA sequencing automation techniques."
Edited by Bruce A. Roe, Judy S. Crabtree and Akbar S. Khan. Department of Chemistry and Biochemistry, The University of Oklahoma.

RNA Analysis
From the Melbourne Signal Transduction Group.

RNA Extraction and Purification Methods
From the Melbourne Signal Transduction Group.

Roe Laboratory Protocols
University of Oklahoma's Advanced Center for Genome Technology - ACGT.

Salk Flow Cytometry Protocols
Salk Institute Flow Cytometry Laboratory.

Spector Laboratory Protocols
Routine protocols from the Spector lab at Cold Spring Harbor.

The Joy of Cloning
Technical Protocols used in the Schimenti Lab.

TIBS Methods and reagents monthly column
Methods and reagents is a unique monthly column.

Transgenic Animal Procedures
From The University of Michigan Transgenic Animal Web.

Veterinary Pathology Pages
Histology Protocols from The University of Bristol.

Company Resource Pages:
CLONTECH User Manuals

Eppendorf Scientific
Applications Online
PCR, electroporation, nucleic acid purification, microinjection and micromanipulation.

The Nest Group
Protocols and Applications Guides
HPLC, electrophoresis and dialysis methods.

QIAGEN handbooks and protocols

Roche Molecular
Molecular Biochemicals Technical Manuals
A variety of Manuals and Brochures to assist in research efforts.

Looking for a Vector? Try These Links:

REBASE - The Restriction Enzyme Database from New England Biolabs

Websites Specializing In Protocols


PCR Jump Station

Manual for Autoclave
(TOMY High Pressure Steam Sterilizer BS 305)
1. The lid closes by Clockwise turn and opens on anticlockwise turn.

2. Open the autoclave only when there is light at the “COMPLETE” or at “READY”.
Check the temperature. It is a must that the temperature must be below 600C to open and remove the autoclaved items from inside. Use gloves for safety.

3. Check the water label. It must be between 2 to 5 mm from the base.
a. if less add water from the tap
b. If more open the front door, there is an outlet at the base. Open it to let the water out. Make sure there is a container ready to collect it.
4. Put the triangular stand.
5. Put the legged netted basket (netted basket with stand) first at the lowest position.
6. Fill the basket till its brim only.
7. Put subsequent basket, firmly hanging on the rim attached to the triangular support.
8. Push the cover to the center. Rotate the circular handle clockwise until tight.
9. Press the “SET” button on the top right corner.

10. Adjust the temperature and Time to required level using UP (▲) and DOWN (▼) arrows.
11. When every thing is ready Press START
12. When it need to be cancelled half way through press STOP and wait for the light to come down to Complete.
13. CHECK button is used to check the time and temperature when the machine is in use without disturbing the working.


Bio Shaker /Bio-Oven (BR-13FP)

1. For incubation
2. To shake the samples continuously during the incubation
The outline of the Bio Shaker used in the laboratory of Plant breeding is given below.

The Bio Shaker is suitable for growing cultures of micro-organisms (bacterial cultures) and other tissue, under controlled conditions of Temperatures and agitation, this shaker provides efficient transfer of Oxygen to the culture and optimum temperature can be set.
This machine can be used in two shaking modes:
1. Rotary and
2. Reciprocal shaking

Temperature range: 15ºC to 60ºC
Shaking speed (r/min) 20 - 300

Steps to operate the machine

1. Switch on the machine.
2. Open the front door using the door knob. Then put the samples on the stand appropriately and close the door.
3. Set the optimum temperature suitable for the samples, the temperature range that can be chosen for this machine is 15ºC to 60 ºC.
4. Set the optimum speed as per the requirement. Generally for bacterial culture, 195 r/min is good enough.
5. Set the time if necessary.
6. Choose the appropriate mode of shaking, rotary mode (circular) or reciprocal (sideways, to and fro) shaking mode.
7. Then push the start button.
8. When ending the use of the shaker, push the stop button. When the shaker stops shaking, carefully remove the samples. Then switch off the machine.

C. CTAB for DNA extraction

DNA extraction (CTAB method)
1. Put the sampled leaf (3g) in 50ml tube together with SDS 0.3g, PVP 0.3g and cooled metal cone.
2. Freeze the tube in liquid nitrogen.
3. Crush the sample by Multi-Beads Shocker (1400rpm, 15s×2).
4. Add the preheat (at 65℃) 2×CTAB 3ml and 1×CTAB up to 20ml, then mix.
5. Add the Proteinase K 500μl (Pro.K 4mg/1×CTAB 10ml).
6. Incubate for 30~60min at 65℃ in water bath (shaking it occasionally).
7. Cool at the room temperature for 30min and add equal volume of chloroform / iso-amyl alcohol (24:1).
8. Shake 60rpm for 30min.
9. Spin 3000rpm for 30min.
10. Remove 10~15ml of supernatant to clean 50ml tube.
11. Add equal volume of isopropanol ,shake gently and precipitate DNA.
12. Remove DNA using blue tip to 1.5ml micro tube.
13. To wash, add 70% ethanol and spin down for 1min.
14. Discard ethanol and add 80% ethanol , then spin down.
15. Wash with 100% ethanol same as above.
16. Dry in incubator and add 1/10TE + RNase 200μl.
17. Incubate for 60min at 58℃ in water bath.
18. Dissolve DNA at incubator overnight.
19. Store at refrigerator.

D. HCl and NaOH

1. The Experimental tool what you need must be washed and dried.

Measure distilled water by graduated cylinder, and move into beaker.

2. Deep hydrochloric acid is measured and taken with another beaker and added to 1 little by little,

while stirred well.

※about concentration

The concentration of marketed deep hydrochloric acid (HCl35%) is 11.33N. so, if you want to make

ⅰ) 1N, you must add 88.5g of deep hydrochloric acid to distilled water and make it 1L.

ⅱ) 5N, you must add 442g of deep hydrochloric acid to distilled water and make it 1L.

ⅲ) 10N, you must add 885g of deep hydrochloric acid to distilled water and make it 1L.

3. Quit stirring, add distilled water till wished volume and stir again.

If needed, store in the reagent pot. Write the reagent name, concentration, date, and maker.


Inside the pot of deep hydrochloric acid is full of toxic HCl gas, and it spouts out when you open the pot.

So be careful not to breathe the gas and be careful to be well ventilated.

Wash down as soon as possible when the reagent sticks to skin or eye.

Be careful for generation of heat when you dilate deep hydrochloric acid. Adjust volume after cooled down.

The concentration of marketed deep hydrochloric acid is estimated one. Titrate by known alkaline when

you want to know the exact concentration.


1. The Experimental tool what you need must be washed and dried.

Measure distilled water by graduated cylinder, and a half of them should be moved into beaker 1, and

the other should be reserved

2. Measure NaOH using a balance and a beaker 2, and add it to the beaker of 1 as soon as possible.

Dissolve beaker2-remaining NaOH by distilled water of graduated cylinder, move it into the beaker and stir up.

NaOH is 40g/mol. So if you want to make

ⅰ) 1N, you must add 40g of NaOH to distilled water and make it 1L.

ⅱ) 5N, you must add 200g of NaOH to distilled water and make it 1L.

ⅲ) 10N, you must add 400g of NaOH to distilled water and make it 1L.

3. If needed, store in the reagent pot. Write the reagent name, concentration, date, and maker.


The granular NaOH deliquesces, or absorb atmospheric water. When you use a balance, put a paper

between a beaker and a scale. Weigh as soon as possible. NaOH pot must be tightly closed at once.

Be careful for generation of heat when you dilate NaOH. Since the steam is toxic, be careful not to

breathe the it and be careful to be well ventilated. Adjust volume after cooled down.

Wash down as soon as possible when the reagent sticks to your skin or eye.

Since the size of a NaOH particle is large, you cannot weigh exactly.

Titrate by known alkaline when you want to know the exact concentration.

The scale must be used carefully. Clean up immediately when you spill any reagents.

E. Instant manual of balance

1. Do confirmation that the foam of a water level is in a circle.
(If the foam not stays in the circle, turn the lever of the side and adjusts it.)
2. Turn on the power supply.

A. Measurement of the weight you want.
3. Put powder paper or container on table, and push the button, TERA/T.
4. Add reagent or sample, until the weight you want.
5. Take out the reagent or sample, and turn off the power supply.

B. Measurement of the weight of samples.
6. Push the button, TERA/T.
7. Put samples on table, and read the graduations.
8. Take out the reagent or sample, and turn off the power supply.

Clean the balance and laboratory tables, after you use.

F. Multi beads shocker Preparation of the sample
1. Put the samples you want to crush in the 2ml tubes or 15ml tubes with screw cap.
2. Add 1 metal corn to 1 tube.
3. Fasten the cap.

 How to change the tube holder and How to use the multi beads shocker
1. Choose the tube holder with 24holes (for 2ml tubes) or 3holes (for 15ml tubes).
2. Open the top, and unfasten the 3 screw nuts with black head (nut A), then unfasten the 6 silver head screw nuts (nut B), and remove the tube holder.
3. Set another tube holder (red point is on this side).
4. Fasten nut B in a diagonal line.
5. Set the sample tubes, and slide upper plate on the tubes.
6. Fasten nut A.
7. Turn on the multi beads shocker. The switch is behind of the machine.
8. Set the timer less than 15seconds, and push the start button.
9. Unfasten nut A, get out the sample tubes.
10. Take off the metal corns in the sample tubes.
11. Soak the metal corns in 1N NaOH.

 How to wash the metal corn
1. Wash them in water without SCAT.
2. Rinse them with distilled water, after that ethanol.
3. Dry them in the incubator.

G. PCR Machine Instant Manual

Chapter 1 Overview of Each Machine -----p
1-1 Introduction ------------------------------p
1-1a Peltier Thermal Cycler -----------p
1-1b PCR System 9700 ------------------p
1-1c Master Cycler ------------------------p
1-2 Functional List --------------------------p

Chapter 2 Instant Manuals ------------------p
2-1 Peltier Thermal Cycler ---------------p
2-2 PCR System 9700 ----------------------p
2-3 Master Cycler ----------------------------p

Chapter 1 Overview of Each Machine

1-1 Introduction

As you know, PCR is one of the most useful ways for the molecular researchers because of its convenience of the method. However, no one can use the same PCR program every time as the amplification condition depends on the primers and the length of fragments amplified. So, every user must learn how to make and edit PCR program and find the best amplification condition in the experiments. There are three types of PCR machines in this laboratory, including Peltier Thermal Cycler, PCR System 9700, and Master Cycler. In this chapter, the features of each machine are explained briefly.

1-1a Peltier Thermal Cycler (MJ)
Mostly, three types of vessels, 8-well tube, 96-well plate, and 384-well plate, are used in PCR. Many PCR machines are designed for 8-well tube and 96-well plate, however, Peltier Thermal Cycler can adapt to all of the three types of the vessels by changing the unit. There are two machines and three units, including two 96-well plate adapters and one 384-well plate adapter in this laboratory. Usually, one machine mounts 96-well plate adapter, and the other mounts 384-well plate adapter. In case, both machines can equip 96-well plate adapter. Moreover, user can create gradient PCR program with Peltier Thermal Cycler. Therefore, this machine can cover almost all of the amplification conditions required in PCR experiments.

1-1b PCR System 9700 (ABI)
No special functions are equipped with PCR System 9700. The available vessels are 8-well tube and 96-well plate. “High stability”, that is the one and all features of PCR System 9700. You can see this name in a lot of literatures. This fact also suggests how many people trust PCR System 9700.

1-1c Master Cycler (eppendorf)
The red-letter feature of Master Cycler is that this machine equips CPU just like PC, followed by multi-function and user-friendly interface. Especially, the heat acceleration of Master Cycler between PCR steps is superior to other machines in this laboratory. But don’t forget to shut down the system before you cut the power for CPU!
1-2 Functional List
* : Conventionally, researchers must add mineral oil into the wells for the reaction mixture would evaporate and liquefy on the cold lid while heating. Heat lid function solved this problem and freed the researchers from this trouble.

H. Protocol for PCR (using ExTaq from TAKARA)

1. Take 10x PCR buffer & dNTP out from the freezer and let them thaw.
2. Put template DNA*1, PCR primers*2, buffers and other solutions necessary for reactions into PCR tubes.
The template DNA(10ng/μl) 1  μl
PCR primers (2,5μM) 2  μl
10xbuffer 1  μl
dNTPs (2μM each) 1  μl
DMSO 0.5 μl
ExTaq DNA polymerase 0.04μl
Sterilized water 4.46μl (up to 10μl)

Usually you can’t scale the 0.04μl ExTaq DNA polymerase, so what you have to do is:
1) Put DNA and Primers into each PCR tube.
2) Mix other solutions and water for all reactions in a 1.5ml eppendorf tube. If you are going to perform 10 reactions, Mix;

10xbuffer 1  μl X 11 = 11 μl
dNTPs (2μM each) 1  μl X 11 = 11 μl
DMSO 0.5 μl X 11 = 5.5 μl
ExTaq DNA polymerase 0.04μl X 11 = 0.44 μl
Sterilized water 4.46μl X 11 = 49.06μl

And then, you put 7μl PCR cocktail into each tube.

*1 The template DNA should be less than 10ng per 10μl final volume of PCR reaction.
*2 You can double the amount of PCR primers to get better results.

3. Start reactions with thermal cycler. The protocol attached to ExTaq says PCR cycle should be:
95℃ for 1min

98℃ for 10sec
58℃ for 30sec X 30~40cycles
72℃ for 1min

72℃ for 3min
But the annealing cycle(58℃ for 30sec) is actually too short for 20mer primers with which we work. You’d better set this time at least 45sec, or make your primers longer than 24 bases.
4. After setting the PCR reactions, you make 1.4% agarose gel for electrophoresis.
Put a beaker on a scale and add:

Agarose 0.7g
0.5xTBE buffer 50ml

Actually gel amount depend on which gel frame you use. Ask anybody in the lab for detail.
Put the beaker in a microwave and heat until all agarose dissolve completely and clear solution is obtained. Stir by shaking the beaker every 30 sec.

5. When PCR reaction is finished, add 1~2μl loading buffer(blue color) to each sample and load into wells.

6. Run the gel with 100V for 20-40min.

7. Check reactions by transilluminater and take pictures.

I Spectrophotometer  Biospec1600

By means of this machine, you can investigate concentration of protein and DNA, purity of DNA, absorbance of samples, spectrum of samples and so on.
[Basic Operation]
Turn on the power and select the mode.
According as the situation, correct of the blank.
Inject the sample to cuvette.
Set the cuvette into the cell and measure with start key.
When you deal with cuvette, don’t touch absorptivity sides of the cuvette.
After use, rinse the cuvette with distilled water, and turn it over drying.
A 蛋白質定量
B DNA/蛋白質
eppendorf recommendation
You can investigate concentration of protein and DNA, and purity of extracted DNA.
Generally, when DNA(260nm)/protein(230nm) indicates 1.8~2.0, the DNA can comparatively be said pure.
C フォトメトリック
It is a easy method applying to that absorbance is proportional to concentration.
1. Configure measurement condition. (wave length etc.)
2. According as the situation, correct of the blank and push AUTO ZERO key.
3. Set the sample and measure with START key.
D スペクトラム
This mode can investigate the spectrum of samples.
1. Assign the range of wave length
2. According as the situation, correct of the blank and push BASE補正 key.
3. Start the measurement with START key.
4. The machine displays the result, so manage your purpose.

eppendorf  Bio Photometer
With this machine, you can easily investigate concentration of each nucleic acid, and purity than Biospec1600.
1. Select the key that you want to measure.
2. Set the blank and push blank key.
3. Set the sample and push sample key.
☆function of correcting dilution
dilution → Set the condition → enter → Set the sample and push sample key.
Then, it displays the concentration before dilution.
☆Function → display results
So you can see all results.

J. TE buffer

試薬ビンに1M Tris-HClおよび0.5M EDTAを入れ,Distilled waterを加える。
1/10 TE bufferはTE bufferを10倍希釈して作る。

Put 1M Tris-HCl and 0.5M EDTA in the reagent bottle, and add distilled water.
Dilute TE buffer by ten times to make 1/10 TE buffer.


Tuesday, July 04, 2006



The simplest criterion used for the characterization of proteins is their solubility in various media. As in all legumes, the bulk of soybean proteins are globulins, characterized by their solubility in salt solutions. The solubility of soybean proteins in water is strongly affected by the pH. Close to 80 % of the protein in raw seeds or unheated meal can be extracted at neutral or alkaline pH. As the acidity is increased, solubility drops rapidly and a minimum is observed at pH 4.2 to 4.6. This is the isoelectric region of soybean proteins taken as a whole.
The pH dependence of solubility is used in the manufacture of isolated soybean protein, whereby defatted, unheated meal is extracted with water at neutral or slightly alkaline pH, and the protein is then precipitated from the filtered extract by acidification to the isoelectric region.
More precise and detailed fractionation of the proteins can be carried-out by techniques such as ultracentrifugation, gel filtration and electrophoresis.
Since the classical work of W. Wolff, it has become customary to characterize the soybean protein fractions by their sedimentation constants.

Four major fractions, known as 2 S, 7 S, 11 S and 15 S have been studied extensively. (S stands for Svedberg units. The numerical coefficient is the characteristic sedimentation constant in water at 20oC. The figures are not exact but nominal. Thus the 11 S globulin has a sedimentation constant of 12.3). The 11 S and 7 S fractions constitute about 70% of the total protein in soybeans. The ratio 11 S/7 S is a varietal characteristic and may vary from 0.5 to 3.

The 2 S fraction consists of low molecular weight polypeptides (in the range of 8000 to 20000 daltons) and comprises the soybean trypsin inhibitors (see below).

The 7 S fraction is highly heterogeneous. Its principal component is beta-conglycinin, a sugar containing globulin with a molecular weight in the order of 150000. The fraction also comprises enzymes (beta-amylase and lipoxygenase) and hemagglutinins (see below ).

The 11 S fraction consists of glycinin, the principal protein of soybeans. Glycinin has a molecular weight of 320000-350000 and is built of 12 sub-units, associated through hydrogen bonding and disulfide bonds. The ability of soy proteins to undergo association-dissociation reactions under known conditions, is related to their functional properties and particularly to their texturization.

The 15 S protein is probably a dimer of glycinin. Conglycinin and glycinin are storage proteins and they are found in the protein bodies within the cells of the cotyledons.


Some of the problems associated with soybean proteins include
(1) presence of anti-nutritional factors such as trypsin inhibitor,
(2) undesirable beany flavor,
(3) elicitation of allergic reactions in susceptible individuals,
(4) poor digestibility of soybean proteins, and
(5) deficiency in sulfur-containing amino acids.


Research Plan 2005

Genetic Transformation of Soybean (Glycine max Merril L) Via Particle Bombardment

The quick and best method to achieve genotype independent transformation in crops is the delivery of foreign DNA directly into regenerative cells. Micro projectile bombardment also known as particle bombardment or biolistics is presently used in researches to produce stably transformed plants in many economically important crops. It employs high velocity metal (gold or tungsten) particles to deliver biologically active DNA into targeted, published results show that embryonic or meristematic tissues are more transformable and are able to regenerate into transgenic plants. Transient gene expression can be observed in differentiated plant tissues, the expression of foreign genes is examined by visual and quantitative methods and the study of gene responsible for plant ontogeny can be possible.


The objective of the research is to improve the protein quality in soybean through particle bombardment.

Materials and Methods

Plant material
Preparation of recombinant Plasmid DNA
Isolation of plasmid DNA
Preparation of genomic DNA from Soybean Plant tissue
Identification, Isolation and cloning of genes responsible for coding beta conglycinin
(7S) and glycinin (11S) proteins
Preparation of recombinant plasmid DNA (Plasmid DNA and gene of Interest)
Somatic embryogenesis of soybean seed, Proliferation of tissue
Histochemical analysis
Selection for transgenic
PCR, Southern Blot hybridization
Development and maturation of embryo,
Desiccation and germination

Monday, July 03, 2006




Credits: http://in.news.yahoo.com/040318/43/2c240.html

Friday, June 30, 2006



I was born and brought up , had my education up to Higher Secondary School (grade 12) in Bhutan. I did my bachelors degree from the Institute of Agriculture and Animal sciences, IAAS, Nepal, taking Plant Breeding as elective subject. Right from my child hood I had interest in science, later in biology and then in genetics and biotechnology. The environment that I was brought up in, poverty in the hills and illiteracy among the people; made me think and work towards improvement of agriculture, increment of production and fighting against diseases, pests and weeds. Later studies made me realize that I can do much more than those. I can go deeper into the subject and try out many things which very few people want to try. I want to work with the genes, genomes and genetic transformations. I had a good influences of teachers, seniors and friends who talked about the wonders of biotechnology. Their talks were the source of my wonderment. They encouraged me to take up plant breeding as elective in the fourth year of my Bachelors (BSc. Agriculture). A key hole opened into my oppertunity. i was able to enter Kyoto University to continue my studies taking Plant Breeding as the major. Now I am working with the genetic transformation of Soybean. The title of my present research is “Improvement of Protein Quality in Soybean through Genetic Transformation”. Ref. Web: http://www.ikushu.kais.kyoto-u.ac.jp/member.html . Although I feel I have chosen a big target I am happy that have choosen it.

During my bachelors there were a few experimental works and even the practical works involved a lot of reading and writing than working. Now I understand through doing what I had learnt through reading. Everything is wonderment to me. I am working well. By March 2007 I will be awarded a masters degree. I expect to graduate with “grade A”.

I want to devote my life into the study and research of genes discovering their hidden mystery. I don’t want to limit my potential and discovery to a region or a country but I want to do a thing globally great. I expect warm suggestions and continuous encouragement from all.

Thank you

For contact:
Graduate school of Agriculture
Laboratory of Plant Breeding
Kyoto University
Kyoto 606 8502Japan
Phone 081 80 5306 4481
Or govindarizal@hotmail.com