Use PCR to prepare the parts
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- 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!
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First, map out your assembly. In this example, three parts, A, B, and C will be assembled and inserted into a Vector.
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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 + 15 bp of "Vector right" top strand
- Reverse Primer: 5'-cacaccaCGTCTCa + 15 bp 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
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Part A Primers
- Forward Primer: 5'-cacaccaCGTCTCa + 4 bp of "Vector left" top strand + 15 bp of "Part A" top strand
Note: For insertion into pSB1A3, "4 bp of vector left" = TAGA
- Reverse Primer: 5'-cacaccaCGTCTCa + 4 bp of "Part B" bottom strand + 15 bp "Part A" bottom strand
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Part B Primers
- Forward Primer: 5'-cacaccaCGTCTCa + 15 bp of "Part B" top strand
- Reverse Primer: 5'-cacaccaCGTCTCa + 4 bp of "Part C" bottom strand + 15 bp "Part B" bottom strand
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Part C Primers
- Forward Primer: 5'-cacaccaCGTCTCa + 15 bp of "Part C" top strand
- Reverse Primer: 5'-cacaccaCGTCTCa + 4 bp of "Vector right" bottom strand + 15 bp "Part C" bottom strand
Note: For insertion into pSB1A3, "4 bp of Vector right bottom strand" = TAGT
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Run separate 50 μL PCR reactions for each part. If you are using plasmid DNA as a template, use no more than 10 ng in order to minimize carry-over into the final bacterial transformation step. Check 5 μL of the reaction on an agarose gel.
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PCR Fragment prep
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Digest the template DNA with DpnI
- Add 1 μL FastDigest DpnI and 5 μL of 10x FastDigest buffer to each PCR reaction. Note: DpnI cuts the methylated template DNA, not the unmethylated PCR product. Incubate at 37°C for 15 min.
Reagent
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Vol.
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DpnI Digest
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PCR reaction |
45.0 μL
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10x FD buffer (Fermentas) |
5.0
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DpnI (Fermentas) |
1.0
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dH2O |
---
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51.0 μL
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- Purify the DNA from the 51 μL reaction using a Zymo clean and Concentrator kit (or similar PCR clean up kit).
Dilute the purified PCR product to 20 fmol/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 * 650 fg/fmol ÷ 1,000,000 fg/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.
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Digestion/ ligation reaction
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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.
Reagent
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Vol.
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Thermal cycling
- [45°C, 2 min.; 16°C 5 min.] x25
- 60°C, 10 min.
- 80°C, 20 min.
- 4°C, ∞
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20 fmol of each DNA part |
up to 7.0 μL
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10x T4 ligase buffer (Promega) |
1.0
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T4 ligase (NEB) |
1.0
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BsmBI |
0.5
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dH2O |
0.5
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10.0 μL
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