BioPhotovoltaics

       

Notebook
Part Construction	Synechocystis Transformation

Restriction digests – promoter J23119

1. Digest 2ug of the DNA. Promoter concentration is 87ng/ul, so we need 23ul of the mini prep.

2. Add 2.5ul of the 10X buffer and 2ul of enzymes (1ul of each enzyme)

3. Digest the promoter with Spe and Pst and label it prom dig.

4. Incubate the restriction digest at 37°C for 2 hours, and then 80°C for 20min to heat kill the enzymes.

5. Clean the Spe/Pst fragment using the PCR cleanup kit.

Restriction digests – K592025, K1172501 and K1172303 and cmpA

1. Add 2ug of DNA to be digested – 13.5ul for K592025, 12.6ul for K1172501 and 9ul for K1172303. Adjust each one to 20 ul with distilled H2O. Add 2.5ul of the 10X buffer and 1ul of each enzyme.

2. Add 01ul of XbaI and 1ul PstI to these preps and label them amil dig, rib dig and opr dig.

3. Incubate the restriction digest at 37°C for 2 hours, and then 80°C for 20min to heat kill the enzymes.

4. Run these fragments on the gel and separate them. From the gel, isolate the fragments containing these parts (shorter fragments on the gel) and CmpA should then be purified using the PCR cleanup kit.

Restriction digests – J04450, empty vector for the new part and cmpA for submission

1. Digest 2ug of the DNA – 17ul of the DNA topped up to 20ul with water.

2. Add 2.5ul of the 10X buffer and 2ul of enzymes (1ul of each enzyme).

3. Digest the empty plasmid with Eco and Spe and label it empty dig.

4. Incubate the restriction digest at 37°C for 2 hours, and then 80°C for 20min to heat kill the enzymes.

5. Run on a gel and extract the larger fragment. For CmpA – digest it with Eco and Spe and cleanup using the PCR cleanup kit.

Gel electrophoresis -Here is the link to the original expereiment. For our experiment we have adjusted the steps very slightly. Firstly we used 1% agarose gel at 100V for 30 minutes instead of the given timings. Also we imputed the SybrSafe dye and dissolved 5ul in 100ml of the gel before it solidified.

Gel extraction - Here is the link to the experiment and we used this kit wherehere is the link to the specific experiment we did.

PCR purification - Here is the link to this experiment we did.

Ligations - Here is the link to this specific experiment we did.

For culturing Synechocystis:

Culture received as a streak on a BG11 + glucose plates. Culture immediately restreaked on another plate under sterile conditions and a liquid stock set up. Liquid stock was set up using 200ml X1 BG11 solution and 5 loops of the original culture from the streak plate. Liquid culture set up under 26oC with 2000 lux.