10/24/18

Objective:

• Test for the presence of phage clusters in lysate of soil sample C using PCR and Gel Electrophoresis.

Calculations:

Total Volume= 35 ml

Density of Et Br= 10μg/μl

For 2% agarose = 0.02 X 35= 0.7g

For 0.5% Et Br= 0.005 x 35 x 10= 1.7 μg

Procedure:

1.  35 ml of TBE water was poured into a flask.
2. 0.7 g of Agarose powder was added to the flask
3. The flask was microwaved until the solution bubbled. the flask was taken out  and shaken. Because clumps of powder still remained in the flask, the flask was microwaved and shaken again until all the powder had dissolved.
4. The solution was allowed to cool until it was warm, at a temperature of about 55°C.
5. 1.7μl of Et Br was added to the flask.
6. The solution was then poured onto a electrophoresis apparatus plate (with two dams and combs) and the gel was allowed to solidify.
7. the comb and dams were carefully removed once the gel solidified.
8. The plate was then placed into the electrophoresis apparatus.
9. The apparatus was filled with TBE water until the gel was completely submerged.
10. 5μl of the DNA ladder was put in one of the wells in the gel.
11. 10 μl of  the contents of the three green centrifuge tubes prepared on 10/22/18 were placed in three wells in the gel.
12. The apparatus was connected to the power source and the voltage was set to 100V.
13. the gel was allowed to rest in this electric field for 45 minutes.
14. After 45 minutes, the gels were removed from the apparatuses and placed into a machine that emits UV light, analyzes and records the readings on the gel.

Analysis and Conclusion

My Sample ( along with lab partners)control

After analysis of the results from the gel electrophoresis, this soil does not seem to contain any phages and therefore a new soil sample will now be required. There may also be a possibility that adjustment may be required due to varying quality of the result due to use of different Taq polymerases. this may also be further tested. the verity of the PCR will also be tested with the use of lysate samples that contain phages for sure