Week 7. Semester 3. Moving Forward!!

Our positive controls are in and working! I would like to thank our mentors Cori and Robin for helping us connect with Dr. Schwacke, who has kindly shipped us new controls! The Legionella team at PC sincerely appreciates the support! 

This week we were able to analyze our positive controls with gel electrophoresis, please see picture below. Now that our positives are up and running we will begin analyzing our samples collected from summer which had previously tested positive for either mip or 16s. This semester however, we will be taking it a step beyond gel analysis and will be extracting the DNA bands from the agar gels to send for sequencing. This past Friday, October 17th, we had a first go at trying the protocol and have our fist couple of tubes waiting until we collect a batch to take to the ASU lab. As always, trying a new protocol was a thrill! I would like to thank the team because although we were all very focused, it was a lot of fun working together! 

Please reference below for more details concerning our protocols. 

Analysis on Agarose Gel Electrophoresis (10-17-15)

1% Agarose gel with 1μL/1% SYBER®Green.

(Cori added 5μL/1% SYBER®Green to 50 mL gel)

Loaded:

Molecular Weight Marker (1 μL)

positive control DNA (2 μL)

loading dye (2 μL)

Sterile Water (3 μL)

Loaded 7μL of control DNA sample mix and 1 μL of molecular weight marker.

Ran Gel at 100 Volts for 45 min in a 1x TAE Buffer solution (490mL dI water +10 mL 50xTAE)

Placed gel in gel doc to analyze banding.

Legionella Control DNA shipped by Otto from Virginia Tech

Lane 1	Lane 2	Lane 3	Lane 4	Lane 5	Lane 6	Lane 7	Lane 8
		mip		MWM		16s

Legionella Control DNA left: mip. Middle: Molecular Weight Marker. Right: 16s

Agarose gel DNA Extraction for sequencing (10-17-15)

Utilized UV lamp to visualize bands. Cut with scalpel and placed in DNAase free tubes.

Extracted DNA from gel as per the Thermo Scientific GeneJET Extraction kit:

Added binding buffer on 1mg (agarose):1μL ratio.

Incubated at 55◦C for ≈12 min (until get dissolved). Inverting at 2 min intervals.

Vortexed for 5-10 seconds.

For mip DNA only, a 1 gel volume of 100% isopropanol was added.

Transferred solution to purification column and centrifuged for 1 min at 13.2 rpm. Flow through was discarded.

Added 100µL of Binding Buffer to column and centrifuged for 1 min at 13.2 rpm. Discarded flow through. And centrifuged column an additional minute.

Transferred column to DNA free centrifuge tube and added 50µL of Elution Buffer. Centrifuged for 1 minute at 13.2 rpm. Column was discarded and DNA stored at -20°C.

Experimental Errors:

Small Agarose particles were found in the gel and removed with an alcohol wiped pair of forceps.

Week 6. Semester 3. New Positive Controls are in!

Unfortunately, we were not able to conduct any experiments this week. But the team met to discuss our next steps in the project and organize our samples that have tested positive in the past. With the old controls we couldn't move forward because there was clear evidence of degradation of our positive control DNA. Dr. Schwacke has shipped the PC team new positive controls! So we can now resume our testing. Later this week we will be meeting again to run a gel with the new positive control DNA to test it and hopefully find nice banding. With satisfactory results we will then re-test our previously positive samples and cut out the band in the gel to send for sequencing to then confirm whether we have Legionella.

Legionella bacteria

Photo courtesy of: lne.edu