10.31.18 Gel Electrophoresis for Claire and Lucy PCR

Rationale: Since the samples from the lysates of Claire and Lucy’s positive samples had been processed by PCR, it is now possible to visualize the results through gel electrophoresis and imaging. Therefore, the next logical step would be to use the gel procedure to do this.

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 Experimental tubes).
9. 5µL of ladder was added to both gels (Well 1)
10. 10µL of each experimental sample was placed in wells 2-4.
11. Power source was attached to apparatus and activated. Let run for 45 minutes.
12. Used imaging to view results.

Observations:

• The ladder seemed quite faint compared with the samples in the wells.
• The samples were not allowed as much time to move down the gel as they were in previous experiments, as the timing worked better for us to stop ours slightly early to correspond with other gels being run.

Results:

• The samples displayed negative results on the imaging tool. There was no banding that was displayed above where the dye ran to on the gel, which would have indicated a positive result.

Henry, Nathan Gel 10-31-189ldnx

Conclusion and Next Steps:

• Since the PCR procedure coupled with Gel electrophoresis yielded yet another negative result on a known positive sample, it can be concluded that there must be something wrong with the procedure being used. This would explain why no positive results had been obtained, and a solution is required before PCR testing can be considered reliable once more. The next step will be to do work in the mini-teams assigned by Lathan. We will be helping Claire achieve a high titer lysate with her plates.