DNA extraction was divided in two parts, extraction Protocol and DNA amplification. For the experiment, Anethum graveolems (Dill) was used for the analysis. DNA extraction has several steps. The first step is Grinding and Cell Lysis. For the initial step, a sample of the plant was grinded in a mortar and pestle. The liquid nitrogen was used during the grinding process in order to break the plant tissue. Lysis buffer (AP1) and RNase were added into the power mixture to facilitate the breakdown of the plant’s cell wall. The resultant mixture was incubated in a water bath at a temperature of 65° C for 10 minutes.
In the second part, Precipitation and Filtration, precipitation buffer (AP2) was added to the sample and incubated in ice for 5 minutes. It was then centrifuged in order to separate the layers. The solution was filtered as QIAshredder. The lysate was then applied and the mixture was again centrifuged for 2 minutes. A binding buffer (AP3) was added to the solution. Ethanol was utilized to bind the DNA. The resulting solution was transferred to a DNeasy column and then centrifuged for 1 minute. Wash buffer (AW) was added to the column to wash the DNA.
A hot aliquot of Elution Buffer (AE) was also added to the column. The mixture was then spun to remove the DNA from the column thus obtaining extracted DNA. DNA amplification was accomplished via Polymerase Chain Reaction (PCR). PCR is a very powerful tool that allows the production of several copies of DNA. There are three steps in PCR. First, denaturation is done at 94°C for 30 seconds to change double stranded DNA to single stranded DNA. This breaks DNA H-bonds. Next is Primer Annealing at 45-60°C for 60 seconds to make H-bonds with complimentary areas of the single stranded DNA.
Lastly, Extension is accomplished at 70°C for 60 seconds. Polymerase is added to the primer to make new complimentary stands using single stranded DNA. DNA SEQUENCING An aliquot of DNA from the last PCR experiment was retrieved. Exosap was added to the tube which contained the PCR product to setup Exosap reaction. The mixture was then placed in a thermal cycler and covered with a heated lid. The Exosap program was then run for 20 min at 38oC, 15 min at 82oC and held at 4oC to get high quality of DNA.
After the completion of the Exosap reaction, the product was set up on ice as two separate sequences – forward master mix and reverse master mix. The forward sequence contained forward primer combined with exosap treated DNA in the tube and the reverse sequence contained reverse primer combined with exosap treated DNA in the tube. The both tubes were placed in thermal cyclers and covered with heated lids. OPTI-Sequence program was then run. The program ran for 15 seconds at 94oC, followed by 35 cycles of 30 seconds at 94oC, 15 seconds at 55oC, 1 minute at 65oC and held at 4oC.