Restriction Digestion, Antarctic Phosphatase, and Ligation

Determine the order of the two parts you will be putting together; the one in front will be referred to as the insert, while the one behind will be referred to as the vector. Both the vector and the insert need to have their own separate tube, at least in the beginning.

Restriction Digest

Insert tube

• 3x molar ratio to vector of DNA
• 4µL buffer
• 0.5 µL enzyme 1
• 0.5µL enzyme 2
• 1.0µL BSA
• ddH₂O up to 35µL

Vector tube

• 1/3 molar ratio to vector of DNA
• 4µL buffer
• 0.5 µL enzyme 1
• 0.5µL enzyme 2
• 1.0µL BSA
• ddH₂O up to 35µL

Put both tubes into the 37°C water bath for 2 hours. After, place them into the 82°C heating block for 20 minutes. This deactivates 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.

Antarctic Phosphatase Protocol

1. To the vector tube, add 5 μL of 10x Antarctic Phosphatase Buffer, 9μL of water, and 1 μL of Antarctic Phosphatase. We do this to prevent the vector from closing up again without any insert.
2. Put the tube into the 37°C water bath for 1 hour. After, place it in the 82°C heating block for 20 minutes.

Ligation Protocol

1. Take the insert out of the freezer, and add 5μL of insert and 7μL of vector to a new tube. Label the rest of each tube as Unligated, put the date on the tube, and stick it in the -20°C freezer in case your ligation/transformation doesn’t work. To the single tube of 12 μL mix, add 4 μL of 5X T4 Ligase Buffer, 1μL of T4 Ligase and 3.5 μL of double distilled water. Let this sit at room temperature overnight.
2. Transform cells.