Plasmid extraction from red colonies (allegedly carrying the ligated plasmid J04450-E0430). Isolation of red colonies from the transformation of J04450-E0422 for further plasmid extraction. Plasmid J04450-E0430 digestion to test its expected size.
Today I did plasmid extraction from the eight colonies I isolated from the transformation of J04450-E0430. I followed Miguel Ángel's protocol. I used a new set of solutions that was recently made by Pablo García and Paulina Alatriste. As it can be seen from the electrophoretic gel, the extraction procedure generated a lot of chromosomal DNA contamination. Nonetheless, as this plasmid extraction will serve only to see if the ligation resulted as expected, it does not matter that much this contamination. Lane #1 contains 1kb DNA ladder, all the other 8 lanes contain the proper plasmid extraction from each colony (e.g. lane #2 contains plasmid extraction from isolated colony #2).
I proceeded to test the size of the plasmid with the ligated bioparts just extracted. As I intend to cut the SpeI site, the linearized plasmid should be approximately of the size of 4 kb (the plasmid pSB1A2 with the biopart E0430 alone is 2945 nt long, and the biopart J04450 is 1091 nt long). The digestion was perform as follows:
H20
3.5 μl
Buffer #4 10x
1 μl
BSA 100x
1 μl
DNA
4 μl
SpeI
0.5 μl
Total
10 μl
The digestion was left at 37°C overnight.
I also checked if colonies appeared from the transformation of the ligation of J04450-E0422. Not as many colonies as the former J04450-E0430 ligation appeared. Some of the colonies were just becoming red, meaning they carry the plasmid with the desired ligation. I isolated 6 of these colonies and put them to grow in a separate petri dish with ampicillin.
Finally, I checked the OD at 750 nm of the Chlamydomonas reinhardtii culture I let growing. It is now at 0.34, I expect it to arrive at 1 this monday, so the anaerobiosis induction should be applied the night from monday to tuesday.
UNAM Genomics Mexico 2011
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