IGEM:Peking/2007/Count-Conjugation-Notebook/2007-7-15

Plasmid Construction

What a tired and aweful day!

1. We've minipreped E0040 B0015 J01060 R0040.
2. Digested E0040 with EcoRI/SpeI and pSB3K3 with EcoRI/XbaI.
   • Use 1ul enzyme, 3ul 10x buffer H(SpeI) or M(XbaI), 15ul [math]\displaystyle{ ddH_{2}O }[/math] and 10ul DNA.
   • Two independent experiment digested for 1h and 2h in 37 degree
3. Ran agarose-gel for E0040 and pSB3K3 digestion product: very bad results. Nothing on the E0040 gel, even marker, because of Yang Yifan's sample adding mistake. No DNA was seen on the pSB3K3 gel, although marker was available. (We knew on the next day that there is no DNA in the pSB3K3 minipreped results.)

But we did learn from this day

1. Examined the instructions and protocols for digestion and agarose gel electrophoresis.
2. Decided that use 2h as the digestion time for plasmid DNA.
3. Decided to change the digestion volume to 20ul: 1ul enzyme, 2ul 10x buffer, 5ul DNA and 10ul [math]\displaystyle{ ddH_{2}O }[/math].
4. Learned how to use the UV gel imager.

Things for tomorrow

1. Re-miniprep E0040 and pSB3K3
2. Ran agarose gel for the rest of the BioBricked we've minipreped.