Week of 09/23/24

 

Introduction

Another transformation attempt was undertaken for P81. Varying amounts of plasmid were utilized again to see if altering the concentration might increase the chances of transformation.


Methods

5 microliters of 220 ng plasmid was used for the first experimental group. 7 microliters of 220 ng for the second and 10 microliters for the third.

Transformation Protocol

1) 1 ml cells OD value of 1 (late log)

2) Pellet

3) Resuspend in 100 microliters of TGY

4) Add 40 microliters of 0.3 CaCl2 mix

5) Ice 15 minutes

6) 30 microliter sample and about 10 microliters of plasmid (where the plasmid is 200-400 ng per microliter for a grand total of around 1 microgram of plasmid DNA)

7) Mix

8) Incubate at 30 degrees Celsius for 90 minutes while inverting every 15 minutes.

9) Add  2 ml of TGY and Incubate for 24 hours

10) Plate cells on TGYA and TGYA CM plates

11) Use Cells mixed with PCR water as a blank


Results

Growth on the control plates and no growth on the experimental plates.


Discussion

That was the final attempt of transformation for P81. There are other more promising avenues to research. While transformation of P81 may still be possible given enough time, in order to utilize my time most efficiently we will now direct our energy elsewhere.


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