The TAs will look for these items in your notebook. Please complete a self-check before 1pm on Thursday December 8th.
|Item||Date in your notebook|
|Soil collection, GPS recorded|
|Initial plaque morphology notes|
|2-3 rounds of purification, with notes on plaque morphology and process|
|Webbed plate protocol & image|
|High titer lysate collection & titer calculation|
|DNA purification protocol|
|DNA concentration and agarose gel results||***AJ will post most recent gels Dec 6th afternoon...sorry it's on my office computer|
|TEM staining protocol|
|Host range testing results- class experiment|
|Host range testing results- second experiment|
|Y2H mating on non-selective media, pic & your observations|
|Y2H selection for diploids on -YT, pic & your observations|
|Y2H selection for protein-protein interactions on -YTH, pic & your observations|
|Post your contribution to Y2H or host range posters|
|Post your final presentation slides|
|Post a link to your bacillus.phagesdb.org phage archiving page!! Congratulations, you made it!|
Restriction digestion table- please copy to your own wiki page and enter the appropriate volumes.
|10X reaction buffer|
|500 ng phage genomic DNA|
Pipette in this order: Water, 10X buffer, BSA, DNA, enzyme
From 9/8/16 class: 7 successful enrichment cultures! Hope you can see your name and spots.
From 9/1/16 class: 19 Successful Enrichment cultures! See if you can see your plates, and the spots on them. A few are really difficult to see, but are there!
Summary Techniques and Data Table: Please copy this table onto your own wiki page. It is your responsibility to notify me or your TA of updates to this table!
Demonstrate competent pipetting skills
|Demonstrate appropriate aseptic technique|
|Soil Sample Collection|
choose: Direct or Enrichment plating
|Identification of plaque|
Purification of plaque
(Number and method used)
Describe final plaque morphology
|Production of web plate||Empirical/ Intuitive|
|Collection of high-titer lysate|
|DNA purification, how many columns did you use?|
|DNA quantification, Concentration/volume:|
Estimated volume collected:
How is your tube labeled?
|Restriction digest, which enzymes cut?|
Submitted Archive Report to bacillus.phagesdb.org or
phagesdb.org (Gordonia phages)
|Insert link here!|
Submitted lysate archive to Dr. Johnson
How is your tube labeled?
Our host bacteria
Instructors guide for media preparation, etc., for Gordonia
Bacillus thuringiensis kurstaki
Samples from Iceland
|6/30/16, Soil from under edge of one lonely plant in a tuft of grass, at the edge of a sulfur vent area, rainy and 50oF|
|6/28/16, Grassy area at top of hill overlooking lagoon, sunny and 55oF|
|Waterfall in east Iceland|
Samples from Poland
|Poznan||52.458737, 16.910597||8/2/16, Grassy area beside apartment complex, sunny and mild, 25oC|
8/4/16, Backyard garden of a house, cloudy, 20oC
8/9/16 Enrichment cultures
Set up five enrichment cultures containing ~50ml TSB, 0.5 ml Btk overnight culture, a few grams of soil.
Grew at 30oC, overnight.
8/10/16 Spot test enrichment cultures
- Prepare plate with lawn of Btk- add 4.5ml top agar to 0.5 ml Btk overnight culture, and pour onto luria agar plate.
- Filter enrichment culture sample using 0.22 um filter, into sterile tube. Serial dilute filtrate to 10-5. Spot 3ul of each onto prepared lawn.
- Grow overnight at 30oC.