Use PCR to prepare the parts

1. Multiple parts can be assembled in one step. Parts and the destination vector should be amplified by PCR. Make sure that none of the parts or the vector have any BsmBI sites!

2. First, map out your assembly. In this example, three parts, A, B, and C will be assembled and inserted into a Vector.

3. Design a pair of primers to add BsmBI sites to the ends of a vector backbone. The "cacacca" before BsmBI is used to help restriction enzyme positioning. The "a" after BsmBI is a spacer that is required to generate a correct 4-base sticky end.

Vector Primers

-Forward Primer: 5'-cacaccaCGTCTCa + 15bp of "Vector right" top strand

-Reverse Primer: 5'-cacaccaCGTCTCa + 15bp of "Vector left" bottom strand

pSB1A3 Vector Primers - already available in the Haynes lab freezer

-Forward Primer gg0001: 5'-cacaccaCGTCTCaactagtagcggccgctgcag

-Reverse Primer gg0002: 5'-cacaccaCGTCTCatctagaagcggccgcgaattcc

4. Create primers for parts as follows:

Part A Primers

Forward Primer: 5'-cacaccaCGTCTCa + 4bp of "Vector left" top strand + 15bp of "Part A" top strand

Note: For insertion into pSB1A3, "4bp of vector left" = TAGA

Reverse Primer: 5'-cacaccaCGTCTCa + 4bp of "Part B" bottom strand + 15bp "Part A" bottom strand

Part B Primers

Forward Primer: 5'-cacaccaCGTCTCa + 15bp of "Part B" top strand

Reverse Primer: 5'-cacaccaCGTCTCa + 4bp of "Part C" bottom strand + 15bp "Part B" bottom strand

Part C Primers

Forward Primer: 5'-cacaccaCGTCTCa + 15bp of "Part C" top strand

Reverse Primer: 5'-cacaccaCGTCTCa + 4bp of "Vector right" bottom strand + 15bp "Part C" bottom strand

Note: For insertion into pSB1A3, "4bp of Vector right bottom strand" = TAGT

5. Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10ng in order to minimize carry-over into the final bacterial transformation step. Check 5 μL of the reaction on an agarose gel. The image below shows what the resulting fragments should look like after PCR prep.

PCR Fragment Prep

6. Digest the template DNA with DpnI

Add 1 μL FastDigest DpnI and 5 μL of 10x FastDigest buffer to each PCR reaction. PCR product. Incubate at 37°C for 15 min.

Note: DpnI cuts the methylated template DNA, not the unmethylated.

7. Purify the DNA from the 51 μL reaction using a Zymo clean and Concentrator kit (or similar PCR clean up kit).

8. Dilute the purified PCR product to 20fmol/μL

-Measure ng/μL of the purified sample.

-The volume of purified DNA (x) you will need to dilute in a final volume of 20 μL = length in bp ÷ measured ng/μL * 20 fmols/μL * 650fg/fmol ÷ 1,000,000fg/ng * 20 μL final volume

-Formula: x = length in bp ÷ measured ng/μL * 0.013 * 20

-Note: final volume can be changed by changing the last number from 20 to something else.

Digestion/Ligation Reaction

9. Perform BsmBI/ T4 ligase mediated assembly

-BsmBI cuts the DNA fragments and creates complementary overhangs.

-Complementary sticky ends anneal via base pairing.

-T4 ligase seals gaps in the phosphodiester DNA backbone.

10. Set up the following master mix:

11. Cycle with the following parameters:

Thermal cycling

[45°C, 2 min.; 16°C 5 min.] x25

60°C, 10 min.

80°C, 20 min.

4°C, ∞

Bacterial Transformation

12. Add total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0mL tube.

13. Incubate on ice for 2 min., heat shock at 42°C for exactly 45 sec., immediately place on ice.

14. Add 800 μL sterile SOC medium.

15. Grow with shaking at 37°C for 30 min.

16. Pellet the cells at top speed in a microcentrifuge for 3 min. at room temp.

17. Discard the supernatant. Resuspend the cells in 100 μL LB + antibiotic.

18. Plate cells on pre-warmed LB agar + antibiotic with sterile glass beads. Grow overnight at 37°C. *Quick-transormation (e.g., DH5α-Turbo) is not recommended