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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.
PCR reaction

• Added the following to each of three PCR tubes: 10 uL 5x Phusion HF buffer, 27.5 uL water, and 1 uL of dNTPs. 5 uL were added of both the forward and reverse primers (at 10 uM), as was 1 uL of the template DNA. The matching of tube numbers, template DNA, and primers was as follows:

Tube #	2	3	4
Template DNA	phsC	phsAB	phsABC
Primers	phsC_F & phsC_R	phsABC_F & phsAB_R	phsABC_F & phsC_R

• Chilled tubes on ice, then added 0.5 uL of Phusion DNA polymerase and put in thermocycler.
• Prior to designing thermocycler protocol, used oligocalc to get the following Tm values for the primers.

Primer	phsABC_R	phsABC_F	phsAB_R	phsC_F
Tm	73˚C	66˚C	64˚C	72˚C

However, these values seem excessively high, so will just start out with an annealing temperature of 55˚C. Devised the following "phs" Thermocycler Protocol
1. Initial Denaturation-- 2 minutes at 98˚C
2. Denaturation--15 seconds at 98˚C
3. Annealing--15 seconds at 55˚C
4. Extension--3 minutes at 72˚C
Steps 2-4 are repeated for 25 cycles 5. Final Extension--10 minutes at 72˚C
6. Hold--indefinitely at 4˚C
Gel of PCR Products
Ran PCR products on an 0.8% agarose gel with 10 uL of ethidium bromide. Loaded 1 uL of each sample with 9 uL of water and 2 uL of loading buffer. Ran gel until done at 60 V, but had difficulty imaging so left aside to deal visualize the next day, unaware that it would spread.

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