We had a team meeting where each iGEM subdivision presented their work for the week.
I shadoewd Vicky wither her Restriction Digest today. Although I did not quite understand her part completely, I tried my best to follow through the procedures and to understand why she was doing what she was doing.
Procedure for RD:
oth the vector and the insert need to have their own separate tube, at least in the beginning. This is important because it allows for clean addition new parts to a the circut
In the Insert Tube...
-600ng of DNA (To figure out the volume, the calculation is 600 / concentration of plasmid. This gives you volume in μL).
-Water, so that the volume of DNA and water in the tube is 35 μL
-4 μL of React 1 Buffer
-0.5 μL of EcoR1
-0.5 μL of Spe1
In the vector Tube...
-250ng of DNA (To figure out the volume, the calculation is 250 / concentration of plasmid. This gives you volume in μL).
-Water, so that the volume of DNA and water in the tube is 35 μL
-4 μL of React 2 Buffer
-0.5 μL of EcoR1
-0.5 μL of Xba1
Put both tubes into the 37°C water bath for one hour. After, place them into the 65°C heating block for 10 minutes. This destroys any enzymes in the tube (which is ok, because by now they’ve done all they need to). Take the insert out, and put it in a -20°C freezer.
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