IGEM:Imperial/2010/Vectors team

Objectives

• Assemble the PyrD vector with the Pme integration site
• Assemble the AmyE vector

Schedule

Week 6

Week 6	Monday	Tuesday	Wednesday	Thursday	Friday
MORNING					Restriction digestion of 5' integration site for the PyrD vector ( Using Spe and Pst)
AFTERNOON				Starting assembly of PyrD vector Annealing the synthesized oligos ( Pme and diff sites)	Gel analysis of resultant products from digestion mixture PCR purification of digestion mixture Further gel analysis

Week 7

Week 6	Monday	Tuesday	Wednesday	Thursday	Friday
MORNING	Starting assembly of AmyE vector Restriction digestion of BB k14070 for the AmyE vector (using Eco and Spe) Restriction digestion of BB k14064 for the AmyE vector (using Eco and Xba) Restriction digestion of 5' integration site (k08) for the PyrD vector (using Eco and Spe) PCR amplification of vector backbone PSB1C3 for the PyrD vector	Gel analysis and extraction of 5' int site for PyrD vector (repetition of step due to absence of DNA during gel analysis yesterday) Gel purification of k70 Gel purification of k08 Re-analysis of k70, k64 (for AmyE) on gel to work out ratios for ligation set up Re-analysis of k08, Pme oligos and pSB1C3 (for PyrD) on gel to work out ratios for ligation set up Restriction digestion of pveg promoter (k53) (using Eco and Spe) and spec cassette (k65) (using Xba and Pst) for PyrD vector Restriction digestion of pSB1C3 for PyrD (using Eco and Pst)
AFTERNOON	PCR purification of PSB1C3 vector PCR purification of BB k14064 digestion products Gel analysis of digestion products of BB k14070 Gel extraction of digestion products of BB k14070	Restriction digestion of k64 and subsequent PCR purification (repetition of step due to absence of DNA during gel analysis) Gel analysis of k70, k64 (for AmyE) to work out ligation ratios Gel analysis and extraction of k53 and k65