Laboratory name
Washington University
Day 1: Setup PCR. Prepare 100 µM stock solutions of the forward and reverse PCR primers in TE buffer. Prepare template DNA stock at a concentration of 10 ng/µl in TE buffer. For a 50 µl PCR reaction mix the following: 25 µl 2X Phusion Master Mix (New England Biolabs Cat. No. F-531L) with 1 µl forward primer, 1 µl reverse primer, 1 µl template DNA, and 22 µl dH2O in a 0.2 ml PCR tube with a dome cap. Be sure to keep the PCR mix on ice at all times. Centrifuge briefly to ensure that all the liquid is at the bottom of the tube. Determine the annealing temperature of the forward and reverse primers according to the method of Breslauer et al., Proc. Nat. Acad. Sci. 83, 3746-50 (1986). A calculator to determine annealing temperature can be found here, ( PCR program: step 1 at 98°C 30 sec, step 2 at 98°C 10 sec, step 3 at 55-72°C (at the annealing temperature determined from primer sequences) for 20 sec, step 4 at 72°C for 1 min/Kb, repeat steps 2 through 4 for 29 cycles, extend at 72°C for 10 minutes to fill in product ends, then hold 4°C. Day 2: Run 1% agarose gel in TAE to verify PCR product size. Dissolve 3 g of ultrapure agarose (Invitrogen Cat. No. 16500-500) in 300 ml TAE. Heat in microwave on medium heat for 4-5 min then add 3 µl Ethidium Bromide solution at 10 mg/ml. Let the solution cool some before casting the gel. When solid, remove gel combs. Load 5 µl PCR product with 2 µl of 5X GelPilot DNA loading dye (Qiagen Cat. No. 239901). Load appropriate DNA fragment ladder to aid in size determination. For example, 6 µl of GelPilot 100 bp Plus DNA ladder (Qiagen Cat. No. 239045). View gel using a UV transilluminator at 302 nm wavelength. For PCR cleanup, follow the QIAquick PCR Purification protocol found on handbook pages 19-20. Briefly, transfer the remaining 45 µl PCR product to a 1.5 ml microcentrifuge tube. Add 5:1 Buffer PB (225 µl), mix by pipetting. Transfer to a QIAquick Spin Column (pink) and centrifuge at 14,000 rpm for 1 min. Add 750 µl Buffer PE, centrifuge at 14,000 rpm for 1 min. Discard ethanol and centrifuge again at 14,000 rpm for 1 min. Transfer column to a clean 1.5ml microcentrifuge tube. Let sit at room temperature for ~20 min. Elute with 50 µl of EB buffer. Trim the ends of the purified PCR product with the appropriate DNA restriction enzymes. For example, for a NheI-XhoI double digestion mix the following: 50 µl PCR product in EB, 20 µl 10X NEB buffer #4, 5 µl NheI, 5 µl XhoI, and 120 µl dH2O (RNAse/DNAse- free). Incubate the reaction at 37°C for 1.5 hours. Recover the trimmed DNA fragment from the digestion reaction using a QIAquick spin column (pink) as described above. Elute with 50 µl EB buffer. Label the tube and store. Prepare pET-21a(+) vector to receive PCR fragments. Digest 2 µg of vector DNA with NheI-XhoI as follows: mix 2 µl DNA at 1 µg/µl, 20 µl 10X NEB buffer #4, 5 µl NheI, 5 µl XhoI at 10 units/µl, and 168 µl dH2O. Incubate the reaction at 37°C for 1.5 hours. Gently mix the reaction tube, add another 2.5 µl NheI and 2.5 µl XhoI enzyme, spin to collect, and then let the digest continue for an additional 1.5 hours. Clean up the digested vector DNA using a QIAquick Spin Column (pink) as described above. Elute the DNA in 50 ul EB. Dephosphorylate the cut vector with Shrimp Alkaline Phosphatase (Roche Cat. No. 11758250). To the 50 µl cut vector in EB, add 10 µl 10X SAP buffer, 48 µl dH20 and 2 µl SAP enzyme. Incubate at 37°C for 30 minutes. Heat kill the SAP at 65°C for 10 minutes. Clean up the SAP treated vector once again using a QIAquick spin column (pink) as described above. Elute the vector with 50 µl EB buffer. Label the tube and store. Visualize the vector and insert concentrations by loading 5 µl of each onto a 1% agarose gel. If needed, precipitate the DNA and resuspend in TE to a concentration of 100 ηg/µl.
Perform ligation using a Quick Ligation Kit (New England Biolabs Cat. No. M2200S). The volume of DNA and insert should be 10 μl before adding 2X Quick Ligation Buffer. The overall concentration of vector plus insert should be between 1-10 µg/ml for efficient ligation. Adjust the Insert to vector ratio to between 2 and 6 for this single insertion. Allow the ligation to proceed at 25°C for 5 minutes. Transform ligation into GC10 competent E. coli cells (Sigma Cat. No. G2919-10X50UL). Thaw the cells on wet ice. Add 1 µl of reaction mixture to cells and gently tap the tube to mix. Place cells on wet ice for 30 minutes. Heat shock cells at 37°C in a water bath for 45 seconds. Return the cells to wet ice for 2 minutes. Add 500 µl of SOC. Shake at 225 rpm and at 37°C for 1 hour. Plate cells on LB agar plates supplemented with 100 µg/ml carbenicillin to obtain well isolated colonies. Incubate plates overnight at 37°C.