Geochemistry & Ecology of Red Mat Systems (GERMS)

Undergraduate Summer Research Program

Week Four – Practice

Genomic DNA Isolation and PCR

Genomic Isolation Day One:

Procedures: We used .1mm zirconium beads added SDS and GTE. Then we added phenol/chloroform. After a spin on the centrifuge, we had three separate layers, the bottom is the beads, the middle is organic material or the phenol/chloroform and the top layer is aqueous. We removed the top layer and added acetone to allow the DNA precipitate.
Mixing the test tube sample by inverting it 30 times.

Genomic Isolation Day Two:

Procedures: Jana extracting off the supernatant
Gel Results: I didn’t get any information from my run on Imperial red layer mat. This meant that either that mat was dead, or I messed up somewhere in the procedure. The past few years, Imperial didn’t have any results for the red layer.

PCR:

Procedures: PCR machine used to amplify your bacteria.
Results: We were able to successfully amplify the amount of bacteria.

Agarose Gel:

Procedures: Filling the wells of the agarose gel
Results: Lane Results: Imperial Green 1-10 were my samples used. 1: 16s 1X 2: Blank 3: 16s 1/10X 4: Blank 5: 16s Neg. 6: Blank 7: DGGE 1X 8: Blank 9: DGGE 1/10X. 10: Blank 11:DGGE neg. 12: Marker

DGGE Procedures:

Procedures: Framing for the DGGE gel as its being poured
Results: DGGE gel Results. 0: Blank 1: Blank 2. Blank 3: R 19 4: H 43 5: C 6: H 7: Jana’s sample 8:Liz’s sample 9. My sample 10: H 31 11: H 39 12: S 10 12- 15: Blank

Set-up: DGGE set up for the pouring of the gel. Starts at the top on the yellow burner and goes down into the frame to set the gel.

DGGE Results:

It would be safe to say that my lane, lane #9 (Imperial green 1/10 x) matched up with known samples 5 being Green GNS and 6 being Cyanobacteria. The last line from the DGGE gel told me that there also could have been Gram Positive bacillus or G-Gamma Proteobacteria.

Plasmid Isolation:

Procedure: We started off with clones labeled Imperial Green 1-10. We then added STET and lysozyme and boiled for 60 seconds. We then precipitated the DNA with sodium acetate and isopropanol and froze for 15 fifteen minutes. Next we added the DNA to smaller tubes and added an enzyme mix containing EcoRI, buffer and water. We then incubated it for 40-60 minutes. We prepared and agarose gel and let it harden. After the gel hardened we loaded it with our samples and ran it at 75 volts.

Results: This information is from the E. Coli clones grown for our use. We then mixed them with our samples from our various sites along with other aspects of the protocol and then added the samples.

Gel Description and Diagram:

Agarose mini-prep gel results: My samples were Imperial 1 – 10, and the samples went in numerical order, respectively. Lanes: 1: Imperial 1 2: Imperial 2 3: Imperial 3 4: Imperial 4 5: Imperial 5 6: Imperial 6 7: Imperial 7 8: Imperial 8 9: Imperial 9 10: Imperial 10 11: Marker 12: Blank What does this tell us? It shows that for my results the only group that could have had a possible insert would be group 10. The rest are negative for inserts to the vectors.

MINI-PREP ANALYZED DATA

Clone #	EcoRI Product(s) #, sizes	Insert?
1	Imperial Green 1	No
2	Imperial Green 2	No
3	Imperial Green 3	No
4	Imperial Green 4	No
5	Imperial Green 5	No
6	Imperial Green 6	No
7	Imperial Green 7	No
8	Imperial Green 8	No
9	Imperial Green 9	No
10	Imperial Green 10	Yes

Discussion:

I started off the week with genomic isolation and running my mat sample for the Imperial red layer. The agarose gel we ran on the second day came back and I had not a single lane with anything in it (expect for the marker). I was discouraged but Danny reassured me by stating that Imperial red layer in the past had done the same thing when ran on the gels. For the rest of the week, I ran my DGGE gel and my mini-prep gel using Imperial green layer. My DGGE gel run was successful and I was easily able to identify some of the organisms dwelling within this green layer at Imperial. They were Green GNS, Cyanobacteria, Gram Positive bacillus and/or G-Gamma Proteobacteria. My plasmid isolation and mini-prep wasn’t as successful and I only found one vector insert in lane 10, which was sample Imperial Green 10. There wasn’t much diversity because I only was able to obtain one insert. This week I gained an ample amount of experience on working with various gels and determining how to interpret them.