Restriction Digest, Antarctic Phosphotase, 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 Protocol

In the Insert Tube...

• 600 ng 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 both DNA and water in the tube is 35 μL total
• 4 μL of React 1 Buffer
• 0.5 μL of EcoRI
• 0.5 μL of SpeI

In the vector Tube...

• 200 ng of DNA (To figure out the volume, the calculation is 250 / concentration of plasmid. This gives you volume in μL).
• Add water, so that the volume of both DNA and water in the tube is 35 μL total
• 4 μL of React 2 Buffer
• 0.5 μL of EcoRI
• 0.5 μL of XbaI

Put both tubes into the 37°C water bath for one hour. 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

To the vector tube, add 5 μL of 10x Antarctic Phosphatase Buffer, 4 μL of water, and 1 μL of Antarctic Phosphatase. We do this to prevent the vector from closing up again without any insert. Put the tube into the 37°C water bath for 30 mins. After, place it in the 82°C heating block for 10 minutes.

Ligation Protocol

Take the insert out of the freezer, and add 5 μL of insert and 5 μL 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 incase your ligation/transformation doesn’t work. To the single tube of 10 μL mix, add 4 μL of T4 Ligase Buffer, 0.5 μL of T4 Ligase and 6 μL of double distilled water. Let this sit at room temperature for at least 60 minutes.

You are now done. If you are going to transform this construction product, add all 21μL to a tube of whichever competent bacteria you're using.