1. PURIFICATION OF rDNA PCR PRODUCT
follow the "Pellet Paint" protocol (Novagen) for purification of the PCR product:

• transfer 30 mL of DNA solution from the PCR tube to an eppendorf tube. Avoid the mineral oil.
• add 20 mL of dH₂O.
• add 2 mL of pellet paint, mix briefly, then add 5 mL of 3 M Na Acetate. Mix again.
• add 110 mL of 100% ethanol and vortex briefly or thoroughly mix. Incubate at room temperature for 5 minutes.
• spin in microfuge at 14,000-15,000 x g for 5 minutes. A pink pellet should be visible at the bottom of the tube.
• CAREFULLY remove the supernatant by drawing off with a P200. Remove as much liquid as safely possible without losing the pellet. Removing the supernatant by inverting the tube and pouring it off is not recommended.
• DNA wash: rinse the pellet with 200 mL of 70 % ethanol, vortex briefly or thoroughly mix. Spin in microfuge at 14,000-15,000 x g for 5 minutes. Make sure you haven't lost the pellet.
• Remove the supernatant with a P200. Rinse the pellet with 200 mL of 100% ethanol, spin again at 14,000-15,000 x g for 5 minutes.
• Remove the final wash. Residual alcohol can be evaporated with the hair dryer.
• Resuspend the pink pellet in 12 mL of dH₂O. Nucleic acids are fully resuspended when the pellet paint is in solution.

2. RESTRICTION DIGEST

a) plasmid pBLU (total digest volume = 30 mL)

• 12 mL of pBLU (0.1 mg/mL)
• 13 mL dH₂O
• 3 mL Promega 10x Buffer MC (Multicore)
• 1 mL BamHI
• 1 mL Eco RI

b) rDNA PCR product (total digest volume = 20 mL)

• 10 mL of rDNA PCR product
• 2 mL Promega 10x Buffer MC
• 6 mL dH2O
• 1 mL BamHI
• 1 mL Eco RI

c) Incubate both at 37' for at least 30 minutes.
 
 

3. ELECTROPHORESIS

a) Plasmid pBLU

• Remove 18 mL of pBLU digest (0.7 mg DNA) from tube and put into new tube for electrophoresis.
• Return the remaining 12 mL of pBLU to the 37' waterbath.
• Add 4 mL of the 6x Tracking Dye (TD) to the 18 mL of pBLU (total volume = 22 mL).
• Load 20 mL on the agarose gel.

b) rDNA PCR product

• Remove 10 mL of PCR product from the tube and put into a new tube for electrophoresis.
• Return the remaining 10 mL to the 37' waterbath.
• Add 6 mL of dH20 and 4 mL of 6x TD to the 10 mL of PCR product.
• Load the entire 20 mL on the gel.

3. LIGATION

• Heat inactivate both digests for 10 minutes at 70'C.
• Use the tube with the rDNA PCR product and add the following:
• 10.5 mL of digested pBLU
• 2.5 mL of Ligase buffer
• 2 mL Ligase
• Total ligation volume = 25 mL
• Label with your initials and leave on the "ligation" rack for incubation overnight.