Molecular Biology Lab Analysis Assignment | Online Assignment

Please complete the highlighted regions on the attached document.

INTRO: You will be completing the data analysis for Module 5 Day 1 (screening NM400 transformants).  I performed this experiment during spring break and have provided images for you to analyze.  There are up to 6 streaks per group tested on both Kan-50 and Amp-50 plates.  The positive control on each plate is MG1655 + pJL148, and the negative control is MG1655.  Remember that pJL148 is a plasmid that contains the ampicillin resistance gene, and ALSO contains the SPA-KAN cassette, so it contains the kanamycin resistance gene as well.

Part 1: Write a lab notebook entry (electronically) describing the procedures performed (by Dr. Warren) in order to create the differential streak plates.  Use the protocol on Blackboard for Module 5; Day 1 as your guide.  Remember all the parts of a lab notebook entry:  informative title, purpose, procedure, etc.

04/05/2020

Screening NM400 E. coli cells for successful transformation and incorporation of SPA-Kan cassette

Purpose: To screen E Coli. cells.

Procedure:

1. Pick and streak up to 8 unknowns on one LB-Kan plate and LB-Amp plate.
2. Pick and streak positive control.
3. Pick and streak negative control.
4. Incubate overnight at 30°C.

Part 2:  Observe your differential streak plates.  Find your group’s images in the Module 5 folder and save the results for your group data.  There should be one image of your transformants streaked on both the kan and amp plates.  Include your images here in your assignment. (Note:  there is a powerpoint with all the pictures, and also the individual images.  For this experiment, I saved plates by streaking multiple groups onto one plate.  So S4 G1-G4 has section 004 groups 1, 2, 3, and 4 struck on it.)  If there were 6 good colonies available then I tested 6.  For a few transformations there are less than 6 because there were fewer transformants on the original transformation plate.

Images of Streak Plate

Analyze the row “S5

Part 3: First, for each of your streaks, semi-quantitate the growth using this scale.

+ = very good growth              +/- = small amount of growth            – = no growth at all

SPA-KAN          AMP

Streak 1:  +                  +/-

Streak 2: +                   +/-

Streak 3: +                   +/-

Streak 4: +                   +/-

Streak 5: +                   +/-

Streak 6: +                   +/-

Then in 1-2 paragraphs, analyze your differential streak plates and discuss the meaning of these results.  Be sure to conclude whether your tested transformants appear to have successfully integrated the SPA-Kan cassette.

Note: When reading the amp plates, remember to compare the growth you see to the controls on that plate – not just the two plates to each other.   Overall, bacterial streaks on the amp plates may appear to have some low amount of growth, but it’s not very significant, as the same low amount of growth can be seen in the negative control. Also, the amp plates were made with 50µg/ml of amp; this could be increased to 100µg/ml in order to inhibit even more growth.  Overall, note the significant difference between the growth on the kan vs amp plates.  What does this suggest to you?

Part 4:  Conclusion.  Which of your transformants (you can number them from left to right) do you think are correctly integrated and should be checked by PCR?  Is there any evidence that any of your transformants were transformed with pJL148 (the original sourcse of the SPA-KAN cassette)?