Team:Yale/Our Project/Notebook/Week 4

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lab notebook: week 4 (6/28 -7/4)

• Monday 6/28--PCR amplification of thiosulfate reductase operon phsABC
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pSB74 transformants
Extensive colony growth was seen on plated transformations of pSB74 into DH5alpha and LE392 from 6/25 but none was visible on BL21 plate. Continued failure of BL21 transformation suggests that the homemade competent BL21 cells being used are somehow faulty.

PCR amplification of phsABC
• Prior to PCR, took aliquots of all primer samples and diluted them first to 100 uM as detailed below by the chart and then again to 10 uM

Primer	phsABC_F	phsABC_R	phsAB_R	phsC_F
Initial Amount	38.56 nm	46.58 nm	29.03 nm	21.94 nm
Volume of water	385.6 uL	465.8 uL	290.3 uL	219.4 uL

• Similarly, diluted the DNA sample to 10 ng DNA/uL. Started with 0.5 uL of miniprepped pSB74 (sample a) at 67.3 ng/uL and added 2.865 uL of water to get the desired concentration.
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