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Team:Paris Bettencourt/Notebook/TB-ception/Thursday 12th September.html
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<br />• Gibson assembly for the new backbones: PCR products from the 10th of September were used (the same PCR reaction was done for the biobrick pSB2C3 backbone with all primers, so each PCR product for the Duet backbones was mixed with the corresponding pSB product. Protocol: 5 µL of each product, 10 µL of the Gibson master mix, 15 min on 50.0. | <br />• Gibson assembly for the new backbones: PCR products from the 10th of September were used (the same PCR reaction was done for the biobrick pSB2C3 backbone with all primers, so each PCR product for the Duet backbones was mixed with the corresponding pSB product. Protocol: 5 µL of each product, 10 µL of the Gibson master mix, 15 min on 50.0. | ||
| + | <br /> | ||
| + | <br />• PCR for RFP - we wanna add biobrick cut sites on the RFP gene from pSB1C3 (Phusion protocol). Primers: gaattcgcggccgcttctagATGGCTTCCTCCGAAGACGT(RFP F), ctgcagcggccgctactagtaGCGATCTACACTAGCACTAT (RFP R). | ||
| + | <br /> | ||
| + | <br />• PCR to biobrick pET, so we could clone RFP in it and put it under the T7 promoter: template is pET21b+TDMH (Phusion protocol). Primers: tactagtagcggccgctgcagGATCCGGCTGCTAACAAAGC (pET R), ctagaagcggccgcgaattcATGTATATCTCCTTCTTAAA (pET F) | ||
| + | <br /> | ||
| + | <br />• miniprep for DB3.1 + pColA-pSB3C5 and for DB3.1 + pCDF-pSB3C5. Then we did colony PCR for the minipreps with the corresponding primers (pDCA/pDCA3 and pDCA/pDCA1) to check if the insert from the Duet vectors is inside. The bends were the right size. | ||
| + | <br /> | ||
| + | <br />• Digestion of TDMH (biobricked) and the linear backbone: | ||
| + | <br /> | ||
| + | For the backbone: 10 µL of the plasmid, 10 µL of the Green buffer, 3 µL for EcoRI and Pstl, 14 µL of dH2O. | ||
| + | <br /> | ||
| + | For TDMH: 10 µL of the pcr product, 4 µL of the Green buffer, 1.5 µL for EcoRI and Pstl, 23 µL od dH2O. | ||
| + | <br /> | ||
| + | <br />• PCR Ligation for biobricking TDMH: dH20 4.5 µL, TDMH 1 µL, linear backbone 3 µL, T4 ligation buffer 1 µL, T4 ligase 0.5 µL; 16.0 fr 1 h. | ||
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Latest revision as of 14:18, 25 September 2013
TB-ception
ASDFThursday 12th September
• linear bacbone PCR: Phusion protocol from the 10th of the September, new PCR program: 98.0 (30 s) - 98.0 (10 s), 58.0 (15 s), 72.0 (1:10) - 72.0 (3 min). This time the PCR worked fine - no unspecific binding; the band was 2 kb long.
• PCR for biobricking TDMH: we designed primers to add biobrick cutsites on our TDMH gene. Template - pET21b+TDMH miniprep (150 ng/µL), primers - TDMH F (gaattcgcggccgcttctagATGATTTCCCTCCGGAAGCC) and TDMH R (ctgcagcggccgctactagtaTCAGTGGTGGTGGTGGTGGT). Program: 98.0 (30 s) - 98.0 (10 s), 62.0 - 58.0 (30 s), 72.0 (30 s) - 72.0 (10 min). It worked, bend size - 700 bp.
• Gibson assembly for the new backbones: PCR products from the 10th of September were used (the same PCR reaction was done for the biobrick pSB2C3 backbone with all primers, so each PCR product for the Duet backbones was mixed with the corresponding pSB product. Protocol: 5 µL of each product, 10 µL of the Gibson master mix, 15 min on 50.0.
• PCR for RFP - we wanna add biobrick cut sites on the RFP gene from pSB1C3 (Phusion protocol). Primers: gaattcgcggccgcttctagATGGCTTCCTCCGAAGACGT(RFP F), ctgcagcggccgctactagtaGCGATCTACACTAGCACTAT (RFP R).
• PCR to biobrick pET, so we could clone RFP in it and put it under the T7 promoter: template is pET21b+TDMH (Phusion protocol). Primers: tactagtagcggccgctgcagGATCCGGCTGCTAACAAAGC (pET R), ctagaagcggccgcgaattcATGTATATCTCCTTCTTAAA (pET F)
• miniprep for DB3.1 + pColA-pSB3C5 and for DB3.1 + pCDF-pSB3C5. Then we did colony PCR for the minipreps with the corresponding primers (pDCA/pDCA3 and pDCA/pDCA1) to check if the insert from the Duet vectors is inside. The bends were the right size.
• Digestion of TDMH (biobricked) and the linear backbone:
For the backbone: 10 µL of the plasmid, 10 µL of the Green buffer, 3 µL for EcoRI and Pstl, 14 µL of dH2O.
For TDMH: 10 µL of the pcr product, 4 µL of the Green buffer, 1.5 µL for EcoRI and Pstl, 23 µL od dH2O.
• PCR Ligation for biobricking TDMH: dH20 4.5 µL, TDMH 1 µL, linear backbone 3 µL, T4 ligation buffer 1 µL, T4 ligase 0.5 µL; 16.0 fr 1 h.
