10.24.28 Gel Electrophoresis Sample E

Rationale: Since sample had been put through the process of PCR, the results of whether or not phage DNA is present can be processed by using gel electrophoresis and matching bands to visualize results. Therefore, it was necessary to use the gel electrophoresis process to determine the presence of phage.

Procedure

1. Added 35mL 1X TBE to two separate Erlenmeyer flasks.
2. Added 0.7g Agarose to both flasks, then heated until bubbles formed. Once bubbles formed, tube was shaken until bubbles disappeared. Repeated until solution was created.
3. Let cool to about 50°C.
4. Added 1.7µL EtBr to both Erlenmeyer flasks. Swirled to mix
5. Poured contents of flasks onto two separate gel apparatuses that contained combs to create holes in gel.
6. Once gel had set, a small amount of TBE buffer was added to the top of both apparatuses. Comb and rubber seals were removed, and gel was set in machine.
7. 1X TBE was poured over the top of the gel and wells until gel was completely submerged.
8. 2.5µL dye was added to each sample (1-3 Negative Controls, 1-3 Positive Controls, 1-3 Experimental, and 1-3 Henry Experimental).
9. 5µL of ladder was added to both gels (well 4 on gel 1 and well 1 on gel 2)
10. 10µL of each negative control sample was added to the left side of the ladder (wells 1-3) on gel 1
11.  10µL of each positive control sample was added to the right side of the ladder (wells 5-8) on gel 1
12. 10µL of each experimental sample was placed in wells 2-4.
13. 10µL of each Henry experimental sample was placed in wells 6-8.
14. Power source was attached to apparatus and activated. Let run for 45 minutes.
15. Used imaging to view results.

Observations:

• The gel surrounding second negative control well was pierced, causing not all of the sample to be placed into the well. There was enough remaining in the well so the effects should not be overly dramatic.
• Gel had a blue tint when it was solid, helpful to know when it is discouraged to touch the gel that contains EtBr.

Results:

• The gels run showed no presence of bands that would indicate phage on both the negative control and experimental wells, but they showed a very present band on one of the positive control wells, a faint band on one of the positive control wells, and no banding on the third positive control.

Conclusions:

• Since no banding was seen in the experimental wells for the fifth soil sample, it is very likely that there is no phage present. There may be a problem with the PCR procedure, as there have been no positive results since the beginning of its use, but it is overwhelmingly likely if that is not the case that there are no phages in soil sample e. The next steps will be obtaining a new soil sample unless a known positive sample is put through the PCR procedure and results display negative. In that case, the sample would need to be redone with an improved PCR procedure.