From 2007.igem.org

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 100 ng in sample (total volume 20 uL)
  • maximize the insert volume
• ligation I: pCinRHSL (vector) + OmpRBS (insert)

pCinRHSL (vector)	3 uL
OmpRBS (insert)	14 uL
10X buffer	2 uL
T4 ligase	1 uL

• ligation II: pOmpC (vector) + TATA_INSA (insert)

pOmpC (vector)	4.5 uL
TATA_INSA (insert)	12.5 uL
10X buffer	2 uL
T4 ligase	1 uL

• ligation III: CinR+HSL+D-term (vector) + TATB_INSB (insert)

CinR+HSL+D-term (vector)	3 uL
TATB_INSB (insert)	14 uL
10X buffer	2 uL
T4 ligase	1 uL