|
One Shade Too Dark --- tale of condensed tannins in faba bean seed coats |
Menu
| Project Information | Introduction | Objective | Hypothesis | Materials / Methods |
| Results and Discussion | Conclusions | Errors and Future Consideration | Acknowledgements | Bibliography |
PCR method
|
PCR (Polymerase
chain reaction) is a molecular biology technique for enzymatically
replicating DNA without using a living organism, such as E. coli or
yeast. The technique allows a small amount of the DNA molecule to be
amplified many times, in an exponential manner. With more DNA available,
analysis is made much easier. PCR is commonly used in medical and
biological research labs for a variety of tasks, such as the detection of
hereditary diseases, the identification of genetic fingerprints, the
diagnosis of infectious diseases, the cloning of genes, and DNA computing.
The PCR process usually consists of a series of twenty to thirty-five cycles. Each cycle consists of three steps: (1) The double-stranded DNA has to be heated to 94-98°C in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: 1-2 minutes up to 5 minutes. (2) After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 1-2 minutes. (3) Finally, the DNA-Polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. This step is called elongation. The elongation temperature depends on the DNA-Polymerase. The time for this step depends both on the DNA-Polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, 1 minute per 1 kbp. A final elongation step is frequently used after the last cycle to ensure that any remaining single stranded DNA is completely copied. This differs from all other elongation steps, only in that it is longer, typically 10-15 minutes. The above information about PCR is from http://en.wikipedia.org/wiki/PCR. The following is the PCR cycles and reaction used in this project.
RAPD
PCR cycle: 1. 94°C for 3 minutes 2. 94°C for 30 seconds 3. 40°C for 30 seconds 4. 72°C for 1 minute 5. Do last 3 steps 35 times 6. 72°C for 10 minutes 7. 4°C
for the rest of the time PCR
reaction mix 2.5 ul 10X PCR buffer (contains magnesium) 0.5 ul dNTPs (10 mM each) 1.0
ul
primer (10 uM)
0.2 ul Taq 1 ul DNA 19.8 ul
Water
The total amount of the PCR mix in each tube is 25 ul. |