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Genetic Diversity among Elite Sorghum Inbred Lines Assessed with Simple Sequence Repeats (SSR)
by ANAS (2003.6.10)

For details , ask Anas (anasyayak@yahoo.com)

At least there are 4 steps for this experiment:
1.	DNA isolation. 
2.	DNA amplification with PCR.
3.	Electrophoresis to see the amplification result.
4.	Data analysis
A. DNA isolation:
Materials and equipment that must be prepared:
1.	Young leave or another part of plants can be used. We can keep the leave in refrigerator (4oC) for 4 days.
2.	A porcelain grind plate (sterilized is better); 1000l micro pipette; micro centrifuge; micro tube; water bath (65oC incubation); ice box; shaker; and vortex.
Chemicals:
1.	DNA Isolation kit (Nucleon PhytoPure-Amersham Life Science) for extraction and purification. Contain of Reagent I, II, and Resin. Can be used for another plant (List of plants can be seen in handbook protocol).
2.	Liquid nitrogen (hazard chemicals, use protector); chloroform high grade - stored at -20oC (carcinogen, harmful, irritant); isopropanol and 70% ethanol high grade - stored at 4oC (highly flammable); TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).
Protocol for DNA isolation:
A.1 Breaking of the cell wall.
1.	Clean the leave and weigh 0.1g (fresh weight). Put in to grind plate.
2.	Add around 3 volumes liquid nitrogen and grind the tissue to yield a free flowing powder.
A.2. Cell Lysis.
3.	Add 600 l Reagent I to grind plate and transfer the liquid in to a tube, ensuring that all the reagents are fully dissolved. 
4.	Add 200 l Reagent II. Invert several times (1 min) until a homogenous mixture is obtained.
5.	Incubate at 65oC for 10 min. and invert every 1 min. 
6.	Place sample on ice for 20 min.
A.3. DNA extraction
7.	Remove tube from ice and add 500 l chloroform.
8.	Add 100 l of Resin and tubes were sealed with parafilm (to prevent from leakage).
9.	Shake on a tilt shaker for 10 min. at room temperature.
10.	Centrifuge at 4000 rpm for 10 min.
11.	Without disturbing the nucleon resin, pipette the upper solution (DNA containing phase) into a fresh tube. 
A.4. DNA precipitation
12.	Add an equal volume of cold isopropanol. Gently invert the tube until DNA precipitates.
13.	Centrifuge at 8000 rpm for 5 min. DNA will precipitate at tube bottom.
14.	Discard the upper liquid and add with 100  l cold 70% ethanol.
15.	Centrifuge at 8000 rpm for 5 min and discard the supernatant.
16.	Open the tube cover and air-dry the DNA pellet for 10 min.
17.	Resuspend the DNA in 100  l TE buffer and store at -20oC.
B. DNA amplification
Materials and equipment that must be prepared:
1.	Template DNA, forward and reverse primers (specific for sorghum especially for amplification of microsatellite).  
2.	PCR machine (Takara PCR).
3.	1000 l, 200  l, 100  l, and 20 l micro pipettes; 1000  l micro tube; PCR tubes, vortex and ice box.
4.	Sterile milliQ water.
Chemicals:
1.	PCR kit (Taq PCR Core Kit for complete PCR setup - Qiagen product, include PCR handbook) stored at -20oC. This PCR kit can be used for another plant. Kit contents:
a.	Taq DNA Polymerase
b.	10x Qiagen PCR buffer
c.	5x Q-solution
d.	MgCl2
e.	dNTP mix (10 mM each of dATP, dCTP, dGTP, and dTTP)
Protocol for DNA amplification:
1.	Thaw 10x Qiagen PCR buffer, dNTP mix and primer solution. Keep the solution on ice after complete thawing. Mix well all solution before use (using vortex). Note: always kept a Taq DNA Polymerase on ice. 
2.	Prepare a master mix (typically contains all of the components needed for PCR except the templete DNA or sorghum DNA) according to table below. Prepare a volume of master mix 10% greater than that required for the total number of PCR assays to be performed.

Component	Volume/reaction	Final concentration
Master mix		
10x Qiagen PCR buffer	2 l	1 x
dNTP mix	0.4 l	200  M of each dNTP
Forward primer 	0.2  l	25 pmol/ l
Reverse primer	0.2  l	25 pmol/ l
Taq DNA Polymerase	0.1  l	2.5 units / reaction
Sterile distilled water	16.1  l	
Templete DNA (sorghum DNA)		
Templete DNA added at step 4	1  l	20 ` 100ng/ l
Total volume	20 l	

3.	Mix gently the master mix by pipetting up and down a few minutes and dispense 19  l into each PCR tube. All PCR tubes kept on ice before placing in the thermal cycler.
4.	Add templete 1 l template DNA to the individual tubes containing master mix. 
5.	Place PCR tubes in a PCR machine (Takara PCR). Thermal cycler condition according to the table below:
Thermal cycler condition: 
Initial denaturation:	3 min 	94oC
3-step cyling		
Denaturation:	1 min	94oC
Annealing:	2 min	depend on melting temperature of a primer. Approximately 5oC below Tm of primers. 
Extention	1 min	72oC
Number of cyles:	35 x	
Final extention:	10 min	72oC
6.	Switch on the PCR and set thermal cycler according to the table above.
C. Electrophoresis
Materials and equipment that must be prepared:
1.	PCR products and GIBCO 1 kb DNA Ladder (mix with bromophenol blue, sucrose and H2O).
2.	Vertical acrylamide electrophoresis equipment (10cm x 12cm).
3.	Power supply (20 mA, 100 V).
4.	LAS-1000UV mini (FujiFilm) connected with Image Reader Software Version 1.01.
5.	1000 l, 200  l, 100  l, and 20 l micro pipettes; micro tip, and an injection to clean the gel wells.
Chemicals:
1.	Stock solution 10x TBE buffer (pH 8.3) contain of: 
a.	Tris 0.89 M
b.	Boric Acid 0.89 M
c.	EDTA 0.03 M
2.	30% acrylamide solution: 29g acrylamide and 1g BIS (hazard chemicals) are dissolved in 100 ml distilled water.
3.	APS (ammonium persulfate) 10% (w/v): 1g APS is dissolved in 10 ml distilled water.
4.	TEMED
5.	Loading buffer (loading dye) (Toyobo loading dye)
6.	Ethidium bromide (mutagenic). Stock solution 10 mg/ml.
Protocol for electrophoresis:
C.1. Preparing glass plates.
1. Take out plates, an edge support rubber, a comb (20 wells), and clips.
2.	One at a time, spray ddH2O on plates and wipe with kimwipe. Spray each with ethanol and wipe clean.
3.	Place an edge support rubber to edges of front plate and put then the back plates on front plate. Gently push the plates to each other to make them tightly adhere with the edge support rubber. Put clips on sides of plates to prevent gel solution dripped out from plate.
C.2. Gel pouring (8% polyacrylamide gel from 30% acrylamide solution)
4.	Make gel solution according to the table below:
Gel solution 8% acrylamide	
30% acrylamide	4 ml
10x TBE	1.5 ml
Distilled water	9.5 ml
Total	15 ml
APS	150  l
TEMED	9  l
5.	Pour the gel solution slowly into a plate - gap. Avoid any bubbles in plate - gap. Insert the comb on the top of plate - gap and push it down.
6.	Let to harden (solidify) for at least 30 minutes under sunlit. This is the best way to solidify the gel, or put under light for 1 hour (approximately).
C.3. Sample loading
7.	Add 4 l loading dye in to each PCR tube (sample) after PCR process. Always kept the samples on ice before load to the gel well.
8.	Take out clips, an edge support rubber and a comb from plate. 
9.	Pour 1x TBE buffer (the running buffer) into the gel tank (half volume) to submerge the gel-plate. Avoid any bubbles in bottom of gel. Add 1xTBE buffer until an appropriate volume to gel tank.
10.	Prior to sample, remove and clean all well using an injection. Rising the well several times with electrophoresis buffer (running buffer) until all wells are clean. Make sure that the entire gel submerged in the running buffer.
11.	Load the first well with 8 ~ 9 l 1kb marker. Avoid using the end (outer) wells if possible because they are the most likely to run aberrantly. 
12.	Fill all wells with 2 l samples. Connect gel tank to the power supply, switch on the power source and run the gel at 20mA-100V. Monitor the progress of the gel by reference to the marker dye. Stop the gel when the bromophenol blue has reach the gel bottom.
13.	Switch off the power supply and take the gel plate outside.
14.	Take out the gel from plate and soak the gel in a 0.5l/ml solution of ethidium bromide for 5-10 min.
15.	Transfer the gel in to water before examining it to remove excess ethidium bromide. 
C.4. Visualization
16.	Put the gel in a LAS-1000UV mini (FujiFilm) dark box. Tight close the dark box cover and ready to exposure. Image can be saved in a file image for analysis process. UV is carcinogenic and must not be allowed to shine on naked skin or eyes. 

D. Data analysis
Materials and equipments.
1.	File image of the gel.
2.	Software to analysis the gel (Science Lab. 2001 - Image Gauge Ver.4.0). This software can automatically recognize lane and band (the peaks region), measure distance the peak region, and measure the molecular weight (bp) for each band using a equation: log(MW) = ax3 + bx2 + cx + d ( based on GIBCO 1kb marker size).
3.	Cluster analysis software and drawing the phylogenetic tree.
(Web - Software: http://aoki2.si.gunma-u.ac.jp/BlackBox/BlackBox.html).

E. Result
(omitted)