NYMU Taipei/Lab Notes/2007 10 4

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* add ligation mixture into competent cell before it entirely melts and mix well
* 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)
* 42 C for 45 sec. and put it into iced water (4 C)
-
* finish within around 5 mins
+
* 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

Latest revision as of 15:13, 4 October 2007

Contents

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 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 buffer2 uL
T4 ligase1 uL
insert:vector1:7
pOmpC (vector, 10 ng/uL)4.5 uL (45 ng)
TATA_INSA (insert, 20 ng/uL)12.5 uL (250 ng)
10X buffer2 uL
T4 ligase1 uL
insert:vector1:5.5
CinR+HSL+D-term (vector, 20 ng/uL)3 uL (60 ng)
TATB_INSB (insert, 30 ng/uL)14 uL (420 ng)
10X buffer2 uL
T4 ligase1 uL
insert:vector1:7

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
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