NYMU Taipei/Lab Notes/2007 9 20

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(Difference between revisions)
Line 4: Line 4:
*** recommended vector mass: 100 ng to 150 ng
*** recommended vector mass: 100 ng to 150 ng
** MI/MV = C * SI/SV
** MI/MV = C * SI/SV
-
***
+
*** MI/MV = C * 1530/3269 =~ C/2
 +
*** assume VI = X, VV = Y, (X*20)/(Y*60) = C/2, X/Y = 3C/2
 +
*** total reaction volume is 20 uL and buffer is 10X
 +
*** for each reaction we need
 +
**** 1X buffer, thus volume of buffer is 20(uL)/10 = 2 (uL)
 +
**** 1 uL T4 DNA ligase (NEB, M0202)
 +
**** X + Y = 20 - (1+2) = 17 = (1 + 1.5C)Y
 +
**** choose C = 4, 7Y = 17, Y = 2.43, X = 14.58

Revision as of 14:41, 20 September 2007

  • (CinR+HSL) + D-term
    • insert (CinR+HSL): 0.02 ug/uL = 20 ng/uL, 1530 bp
    • vector (D-term): 0.06 ug/uL = 60 ng/uL, 3269 bp
      • recommended vector mass: 100 ng to 150 ng
    • MI/MV = C * SI/SV
      • MI/MV = C * 1530/3269 =~ C/2
      • assume VI = X, VV = Y, (X*20)/(Y*60) = C/2, X/Y = 3C/2
      • total reaction volume is 20 uL and buffer is 10X
      • for each reaction we need
        • 1X buffer, thus volume of buffer is 20(uL)/10 = 2 (uL)
        • 1 uL T4 DNA ligase (NEB, M0202)
        • X + Y = 20 - (1+2) = 17 = (1 + 1.5C)Y
        • choose C = 4, 7Y = 17, Y = 2.43, X = 14.58
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