Restriction digest mix: Restriction digest mix:

Digest four tubes at 37C for 1.5hrs.

Add Gel Loading Dye Purple to digestions. This dye will have a pink dye migrate at 400bp and a blue dye migrate at 4000bp. Electrophorese DNA out on a 1% TAE agarose gel containing SybrSafe DNA Gel Stain. Load 0.5ug of Thermo GeneRuler 1Kb Plus DNA ladder to the first well, then load the DNA into subsequent wells. Electrophorese gel at 120V for ~35min.

DNA fragments look exactly as predicted. Success! Now we will ligate these inserts together to make Ars2.0. To extract the DNA fragments we want out of the gel, we inserted filter paper backed by a dialysis membrane below each desired fragment. The DNA will migrate into the filter paper, and be stopped by the dialysis membrane which won’t allow molecules bigger than 8kDa past it. We checked the migration of DNA using the UV imager (gel on right; visible bands are the vector backbone).

Then we removed the filter paper from the gel and placed each one in a 0.5mL microcentrifuge tube that had a hole pierced in the bottom. We nested these tubes inside 1.5mL microcentrifuge tubes and centrifuged them briefly to force the buffer containing DNA out of the filter paper. We now have the desired fragments cut with complementary enzymes that will let us join these two Parts together with the previously digested pSB1C3.

First, we ligated the two Parts together. Because PArsRGFP is twice as big as PcArsR, we used twice as much volume to approximate an equal molar ratio. Incubate Ligation Step 1 at 16C for 1hr. Then we will prepare a ligation to add the backbone vector. For Ligation Step 2, we will make a negative control of adding water instead of insert.

Ligation Step 1 mix: lllllllllllllllllllllllllllllllllllll Ligation Step 2 mix:

For Ligation Step 2, make the entire MM to ligate with water. Leave 15uL for the negative control, and then take 5uL of MM and add to the Ligation Step 1. Ligate at 16C overnight.