User:Stephanie P T. Yiu/Notebook/Restriction Enzyme Activities at 4Â°C, 37Â°C and 65Â°C/Entry Base
Restriction Enzyme Activities at 4oC, 37oC and 65oC
- Steven Fu
- Tiffany Mak
- Tommy Tam
- Yiu, Stephanie P.T.
Restriction enzyme is an enzyme that cuts DNA at the restriction site of the sequence. As the activity of restriction enzyme depends on the temperature of its environment, the enzyme only cuts the sugar-phosphate backbone at the restriction site during restriction digestion at a temperature. Under unsuitable temperature, the restriction enzyme will lose its biological activity and may even denature. Restriction enzymes XbaI and PstI-HF are restriction sites frequently used in iGEM for biobricks, their efficiency is directly related to the experimental result. Therefore, we would like to investigate how efficient restriction enzymes XbaI and PstI-HF are under temperature 4℃, 37℃ and 65 ℃.
Methods and Materials
For this experiment, pSB1C3-BBa_K516030 plasmid obtained from iGEM Kit Plate 2013 was selected as the digestion assay sample. 1 μl of the sample was transformed into E. coli, inoculated and allowed to grow overnight. The plasmids was extracted from E. coli and purified using alkaline lysis miniprep. One positive experimental setup and one negative control were prepared for each reaction environment, i.e. 4°C, 37°C and 65°C.
Both positive and negative setups contained 300 ng of pSB1C3-BBa_K516030. XbaI and PstI-HF were used in digestion and were not added in the negative control. Instead, it was compensated by an additional 0.4 μl ddH2O to keep the total digestion volume at 18 μl in accordance to protocol. One set of both positive setup and negative control was placed in refrigerator at 4°C. The other two sets were incubated at 37°C and 65°C respectively.
After 1 hour of digestion, gel electrophoresis was carried out to inspect the digestion result.
In Figure 1, both sets show that only the experiment carried out at 37°C shows both the target band cut: 864 base pairs and the remaining band: 2070 base pairs while the other 2 temperatures do not. There is a duller band at 2070 base pairs in the result of 4°C in set 2. But the brightest bands appear in 37°C in both sets. 37°C is the most efficient temperature for restriction enzymes: XbaI and PstI among the three temperatures. Also, bands of set 1 are brighter than that in set 2. This may be caused by lower DNA concentration in set 2.
There are 2 sets of set up, prepared with the same protocol. For the first set, the undigested pSB1C3-BBa_K516030 gives 1 band, while the digested gives 2 bands in Gel electrophoresis, if both XbaI and PstI-HF cuts the DNA sample, the first band is the backbone of the plasmid with 2070bp, and the second is the gene of interest with 864bp. The first, third and fifth lanes to the right of the DNA ladder are loaded with BBa_K516030 incubated with enzymes XbaI and PstI-HF at 4℃, 37℃ and 65℃ respectively; the second, fourth and sixth lanes are loaded with BBa_K516030 incubated without enzymes XbaI and PstI- HF at 4℃, 37℃ and 65℃ respectively. The results are as expected. All lanes except the third lane yield one band only. The third lane yields 2 bands at different band lengths from the single band of the other lanes. Our results show that the enzymes XbaI and PstI-HF work most efficiently at 37℃ among 4℃, 37℃ and 65℃. For the second set, the undigested psB1C3-BBa_K516030 also gives 1 band, while the digested gives more then 1 band in Gel electrophoresis. The sixth, fourth and second lanes to the left of the DNA ladder are loaded with BBa_K516030 incubated with enzymes XbaI and PstI-HF at 4℃, 37℃ and 65℃ respectively; the fifth, third and first lanes are loaded with BBa_K516030 incubated without enzymes XbaI and PstI- HF at 4℃, 37℃ and 65℃ respectively. Yet, unexpected results are shown. All lanes except the fourth and sixth lane to the left of the DNA ladder yields 1 band only. The fourth lane yields three lines at different band length while the sixth lane yields two lines, also at different band lengths. One explanation for the strange difference between the two set up’s results is that there is human error in the second set up. For the fourth lane to the left of the DNA ladder, it is shown that the DNA is not completely digested. There might be some pipetting error and uneven mixing when loading of enzymes.
To minimize the chances of having different results and human errors, pipette slower when loading enzyme into the DNA sample to make sure the required amount of enzyme is really emptied from the pipette tip. Also, before running gel electrophoresis, pipette up and down the set up to mix well.
Both XbaI and PstI-HF can perform their digestive function on plasmids, functioning at best in an environment at 37℃ among 4℃, 37℃ and 65℃.