Week of 09/09/24

 

Introduction
This week, another attempt was made to transform Deinococcus aquaticus P81. 

Methods

We packed the cells 3x this week to see if an increase in cell concentration would help the transformation process. 
We also prepared some new 0.3M calcium chloride.

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 upon the control plates and a lack of growth upon any chloramphenicol plate. 

Discussion
Many avenues were explored this week. We increased cell concentration and we even added chloramphenicol to the remainder of the vials (in which the transformation was supposed to have taken place) after the experiment. After going through the proper experimental procedure, we took the excess from the 15ml conical vials and plated that as well to no avail. The transformation procedure is either unsuccessful or the genetic expression of the resistance genes within PRAD1 is insufficient for the cells to grow on 3 microgram per milliliter cm plates.

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