Japan International Research Center for Agricultural Sciences | JIRCAS

Yam variety identification toolkit: Step 3

Yam Variety Indentification ToolKit
About ToolKit Minimum SSR Marker Finder Inquiry form

Step 3

Step 3: Conduct PCR with nominated markers in step 2, and gel electrophoresis

 

Polymerase chain reaction (PCR) and Gel electrophoresis are common laboratory techniques. In the Yam Variety Identification Toolkit, we use these common techniques, with certain modification to suit to your use in smaller lab condition.

Polymerase chain reaction (PCR)

PCR is basically to multiply copied DNA fragments using “oligonucleotide primers” specially designed specifically for the DNA region of interest (DNA marker).

 

We provide here simple manual for PCR and the list of chemicals and consumables for your convenience.

 

 PCR manual (PDF)

 

If you want to learn more basics, there are various textbooks and useful web sites and are available, for example;

Textbook:

  1) DNA fingerprinting in plants: principles, methods, and applications (2nd edn).

     by Weising K, Nybom H, Wolff K, Kahl G. 2005. CRC Press. ISBN: 0-8493-1488-7

  2) Short protocols in molecular biology (3rd edn).

     by Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith and Kevin Struhl. 1995, Wiley.

     ISBN 0-471-13781-2

 

Web site:

Khan Academy: Polymerase chain reaction (PCR)  

 1) Text

 2) Movie

Gel electrophoresis

Gel electrophoresis to visualize the difference of multiplied copies of DNA fragments by separating DNA fragments based on their fragment size.

 

We provide here simple manual for Gel electrophoresis and the list of chemicals and consumables for your conveience.

 

  Gel electrophoresis manual (PDF)

 

If you want to learn more basics, there are various textbooks and useful web sites and are available, for example;

Textbook:

 1) DNA fingerprinting in plants: principles, methods, and applications (2nd edn).

    by Weising K, Nybom H, Wolff K, Kahl G. 2005. CRC Press. 

    ISBN: 0-8493-1488-7

 2) Short protocols in molecular biology (3rd edn).

    by Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J.G. Seidman, John A. Smith and Kevin Struhl. 1995. Wiley.

    ISBN 0-471-13781-2

 3) Microsatellites: Methods and Protocols. Methods in Molecular Biology vol. 1006

    Stella K. Kantartzi (Ed.). 2013 Springer.

    ISBN: 978-1-62703-388-6

 

Web site:

Khan Academy: Gel electrophoresis 

 1) Text

 2) Movie

Tips: Why polyacrylamide gel electrophoresis?

TThe toolkit recommends polyacrylamide gel electrophoresis. Though Agarose gel electrophoresis remains the most widely used technique for separating DNA fragments, polyacrylamide gel electrophoresis can provide high resolution of DNA molecules such as 10–1,000 bp long. Sometimes the difference of allele size between two varieties/lines are only a few bp, though the variation of allele size of each markers is wide. To detect the difference accurately, the toolkit recommends polyacrylamide gel electrophoresis.

Ready?

If you have identified the best marker set, let us move to Step4.

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