NYMU Taipei/Lab Notes/2007 10 4

From 2007.igem.org

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

NYMU Taipei 20071004 CinR-HSL-D-term,pCinRHSL,pOmpC digestion check.jpg
  • 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

NYMU Taipei 20071004 3 vectors 3 inserts digestion check.jpg
  • 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) 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 buffer2 uL
T4 ligase1 uL
pOmpC (vector)4.5 uL
TATA_INSA (insert)12.5 uL
10X buffer2 uL
T4 ligase1 uL
CinR+HSL+D-term (vector)3 uL
TATB_INSB (insert)14 uL
10X buffer2 uL
T4 ligase1 uL