From 2007.igem.org

Contents 1 digestion check of CinR+HSL+D-term,pCinRHSL and pOmpC (vectors) 2 Re-check the concentration of inserts and vectors 3 ligation setup 4 transformation setup 5 Next tasks

digestion check of CinR+HSL+D-term,pCinRHSL and pOmpC (vectors)

lane A: 1kb ladder lane B: CinR+HSL+D-term x 2 lane C: 1kb ladder lane D: pCinRHSL x 2 lane E: pOmpC x 2

• after gel separation, their O.D. was checked. However, it is too low

Re-check the concentration of inserts and vectors

lane A: 1kb ladder lane B: pCinRHSL lane C: pOmpC lane D: CinR+HSL+D-term lane E: 100bp ladder lane F: TATA_INSA lane G: TATB_INSB lane H: OmpRBS

Owing to concentrations of vectors are too low and A260/A280 ratios are not correct (maybe too much ions) thus, we checked the vectors with inserts by PCR to decide how to ligate them

ligation setup

• criteria
  • make vector have around 100 ng in sample (total volume 20 uL)
  • maximize the insert volume, and insert is larger than vector

ligation I: pCinRHSL (vector) + OmpRBS (insert)	ligation II: pOmpC (vector) + TATA_INSA (insert)	ligation III: CinR+HSL+D-term (vector) + TATB_INSB (insert)
pCinRHSL (vector, 20 ng/uL) 3 uL (60 ng) OmpRBS (insert, 30 ng/uL) 14 uL (420 ng) 10X buffer 2 uL T4 ligase 1 uL insert:vector 1:7	pOmpC (vector, 10 ng/uL) 4.5 uL (45 ng) TATA_INSA (insert, 20 ng/uL) 12.5 uL (250 ng) 10X buffer 2 uL T4 ligase 1 uL insert:vector 1:5.5	CinR+HSL+D-term (vector, 20 ng/uL) 3 uL (60 ng) TATB_INSB (insert, 30 ng/uL) 14 uL (420 ng) 10X buffer 2 uL T4 ligase 1 uL insert:vector 1:7

transformation setup

• extract 4 uL from ligation mixture (total 20 uL)
• use whole competent cell (total 1 mL) for each ligation
• add ligation mixture into competent cell before it entirely melts and mix well
• 42 C for 45 sec. and put it into iced water (4 C)
• finish within around 5 mins (don't put the competent cell in iced water more than 5 mins)

Next tasks

• check the ligation by PCR
  • if the size is correct, perform plasmid extraction
  • else, ligate them again