PHAGE PRESENTATIONEmilee Plautz

INTRODUCTION...

METHODS…Aseptic technique

Soil enrichment cultures

Harvest Enrichment

Spot test/streak plate

Plaque purification(titration/dilution)Phage titer

Harvest MTL

Set up 10 plates (web plates)

Harvest HTL

DNA Extraction

Quantify DNA

Prepare samples of electron microscopy

Analyze EM results

DNA electrophoresis

Phage # 2

● Possible mix up/contamination● After first titer count (3.66x10^8), no

more use

Phage # 1 (FerbieJr.)

● Used as permanent phage for testing

● Had large orange risen contaminant

● Titer: 2.13x10^8● Did not run second digestion

DNA not very prominent or defined. Perhaps not enough DNA to cause gel to light up. First suspicions of DNA potentially dying.

Issue with the ladder, however, most DNA and Enzyme combination did not turn out ideal. Most runs are blurry and too dark or insignificant to notice. Another big indicator that there was not enough DNA.

Spectophometer reading:Initial-260: 0.089

280: 0.039Second- 260: 0.070

280: 0.037

Gel Electrophoresis and Spectophometer

Each green line represent 100nm given by the measurement in the lower right corner. The phage appears to be approximately 40nm wide at the head and a little over 75-85nm long from bottom of the head to the bottom of the tail.

In Conclusion

Due to inconsistent data and unclear images, my phage sample should not be submitted for further testing/imaging. Some of the tests came back inconclusive which led to running the same tests more than once. Due to multiple contaminants there is a possibility of an impure phage. Based on all of the inconsistency of this phage and the experiments ran, again I believe that my phage should not be considered when choosing phages for sequencing.

When comparing my DNA with the Sadalgo genome sequenced on phagesdb.org, my DNA does not cut the same as Sadalgo. In all honesty, my DNA will most likely not cut like any of the other sequenced genomes possibly due to the small amount of DNA causing the images to not come out as clearly as one would like.