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Table1. The RNA was quantified by nanodrop spectrophotometer. Sample L is light treated for 4 hours and D is dark treated. The table shows ratio of absorbance at 260nm and 280nm. The table below shows the isolated RNA amount in ng/µl .
Ananlysis of total RNA on agarose gel
The table shows that absorbance ratio of absorbance is approximately found to be same. From the figure of gel it can be seen that no band is observed in the wells in which light treated samples were loaded. This might be because of experimental error while loading the samples or might be because RNase degraded the RNA. Thus RNase might have contaminated the early step of sample preparation. We noted 2 distinct bands in dark treated samples, which have approximate size of 750bp and 1000bp. These bands might be of single stranded RNA, which is the most abundant r RNA of Arabidopsis thaliana. We assume that band at 1000bp might corresponds to the secondary structure of RNA.
The resolution at 37 °C for dark treated sample is better than that of sample kept on ice. This is because higher temperature breaks the secondary structure of RNA and also prevents the slow migration. In this case the background is clear, but in the ice treated sample we can see some white unclear background. We also see some light bands on top of the gel near the wells due to the genomic DNA. Thus always genomic DNA will be present as contaminant.
All the combination of 6 primers, RT/PCR reaction was carried out at different temperatures. The figure below shows the negative control NORT, Samples run at 53° and 60°.
From the image of gel we can see that some of the PCR reaction were specific and some are unspecific. Unspecificity is may be due to the heating of the sample while mixing or due to amplification during the waiting time. Even the salt concentration in the buffer can be the reason. It was noted that sizes of the unspecific products were small.
No specific product was formed with Sc and the genomic DNA and this unspecificity was more at high temperature. The band formed in well 6 and 7, 12 and 13, 18 and19 were might be because of amplification of antisense primer. The negative control gives band with some of the primers and Sa and ASb was more unspecific. No product was formed in well 3 and 5.
At 53 °C many unspecific products were formed these products may be genomic or cDNA products. This is as expected since the temperature is too low for the primers to bind specifically. In 60 ° C the Sb- ASb and Sb-ASc primer pairs were highly unspecific. This unspecificity might be due to strong 3' end of Sb primer.
The pair Sc-ASb gives a very clear sharp band of product with expected size. So we can say that they are best primer pairs for specific amplification. Primer dimmers appear as light bands at the bottom of the gel. As expected ASc has formed primer dimer in all the combinations because it interacts with itself.
Real time RT-PCR
The data obtained from real time RT-PCR was analysed in the Rotor gene software.
Figure1. Shows the melting curve of 3 light and 3 dark treated samples and negative control.
Figure2. Accumilation curve at 72°C cycle A
Figure3. Accumilation curve at 81°C cycle B
Figure 4 . Standard curve for cycle A
Figure5. Standard curve for cycle B
Figure 7. Comparative curve cycle B .
Table 2. comparative quantitative analysis for cycleA. Std 250
A5 L NoRT
A5 D NoRT
Figure 8. Melting curve for all groups data