Week of 09/16/24

 


Introduction

The transformation procedure was attempted again this week with a particular interest upon the OD value. We are attempting to perfect the procedure in every conceivable way so that any failure for P81 to transform can not be attributed to human error. Additionally, varying amounts of plasmid were used across experimental groups to test whether plasmid concentration has any effect.


Methods


A 278 ng per microliter plasmid extraction was used. The first experimental group was given 4 microliters while the second experimental group was given 8 microliters. The final experimental group was given 12 microliters of a 220.4 ng per microliter plasmid extraction. 

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

An OD value of 1.24 was obtained. Cell growth on the appropriate control plates and a lack of cell growth on experimental plates. 


Discussion

A great OD value was utilized as well as varying concentrations of the plasmid. Changing the concentration has had no effect on the outcome. The experimental procedure was pristine with no apparent missteps to correct. 


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