RAPD (PCR) Amplification
By Berisso Kebede, Department of Molecular Genetics, Institute for Agronomy and Plant Breeding, Univesity of Goettingen, Germany
RAPD:- The development of DNA based markers both PCR based and non PCR based markers have increased our understanding genetic variations. (Random Amplified Polymorphism DNA) is one of dominant markers used in the study of molecular genetics and the assay does not require any sequence information about the species. It can help to identify and specify genetic background of many plant species. This can help to follow up the pedigree of the species and analyse quantitative traits. The method is also used to identify genetic loci for identifying the gene of interest for which the other molecular markers aren�t able to locate due to lack of primer combinations.
Although RAPD markers are useful tools in the study of molecular genetics, technical problems such as unrepeatability of the marker even within the same laboratory facilities are often happen makeing it difficult to produce consistnt result. The procedure is not yet optimized and the method is also not standardized making it difficult for initial users. Therefore before starting the RAPD analysis, many researchers will try to optimize his own method under the exisiting laboratory facility and situation, which consumes much more time. Here is the protocol I am using after repeated optimization of the componenets.
| Product | Stock concentration | Unit | Final concentration | Unit | Volume/tube | Unit |
| DNA | 25 | ng | 50 | ng | 2 | �l |
| Primer | 10 | �M | 0,4 | �M | 1's | �l |
| dNTP's | 10 | mM | 0.2 | mM | 0.5 | �l |
| Taq.Pol. | 5 | U/�l | 1 | U/�l | 0.2 | �l |
| PCR buffer MgCl free | 10x | U | 1x | U | 2.5 | �l |
| MgCl2 | 25 | mM | 3 | mM | 3 | �l |
| H2O | - | - | - | - | 15.8 | �l |
| Total | - | - | - | - | 25 | �l |
Thermal cycler
94oC---------30 sec.
92oC---------60 sec.
35oC---------60 sec.
72oC---------120 sec.
45 cycles
Final extension on 72oC for 5 minute
Gel prepration
Agarose + TAE (1.5%):- 3.5 gm Agarose and add 200 ml TAE then mix until the golden form Agar is diluted in TAE
Load the sample in the gel in TBA or TAE solution using 90 to 120 volts (Run for 3 to 5 hours but it depends on the size of the band one looks for)
Wash the gel with Ethdium Bromide and immerse in water bath to fix the bands
Take the picture of the gel using UV light and camera and analyse the band intensity using the stanadrd size band