NYMU Taipei/Lab Notes/2007 9 20
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| - | * (CinR+HSL) + D-term | + | * (CinR+HSL D. ES #4) + D-term |
** insert (CinR+HSL): 0.02 ug/uL = 20 ng/uL, 1530 bp | ** insert (CinR+HSL): 0.02 ug/uL = 20 ng/uL, 1530 bp | ||
** vector (D-term): 0.06 ug/uL = 60 ng/uL, 3269 bp | ** vector (D-term): 0.06 ug/uL = 60 ng/uL, 3269 bp | ||
*** 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 ([http://www.neb.com/nebecomm/products/productM0202.asp NEB, M0202]) | ||
| + | **** X + Y = 20 - (1+2) = 17 = (1 + 1.5C)Y | ||
| + | **** choose C = 4, 7Y = 17, Y = 2.43, X = 6Y = 14.58 | ||
| + | **** approximately Y = 3, X = 14, C = 3.3 | ||
Latest revision as of 15:37, 20 September 2007
- (CinR+HSL D. ES #4) + 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 ([http://www.neb.com/nebecomm/products/productM0202.asp NEB, M0202])
- X + Y = 20 - (1+2) = 17 = (1 + 1.5C)Y
- choose C = 4, 7Y = 17, Y = 2.43, X = 6Y = 14.58
- approximately Y = 3, X = 14, C = 3.3
