NYMU Taipei/Lab Notes/2007 8 27
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(→plasmid concentration after extraction) |
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- | + | == plasmid concentration after extraction == | |
{| style="color:green;background-color:#ffffcc;" cellpadding="20" cellspacing="0" border="1" align="center" | {| style="color:green;background-color:#ffffcc;" cellpadding="20" cellspacing="0" border="1" align="center" | ||
!part | !part | ||
!concentration (ug/ul) | !concentration (ug/ul) | ||
+ | !A260 (OD) | ||
|- | |- | ||
|pOmpC | |pOmpC | ||
- | | | + | |0.06 |
+ | |0.006 | ||
|- | |- | ||
|EYFP | |EYFP | ||
- | | | + | |0.3 |
+ | |0.030 | ||
|- | |- | ||
|ECFP | |ECFP | ||
- | |23 | + | |0.23 |
+ | |0.023 | ||
|- | |- | ||
|CinR | |CinR | ||
- | |15 | + | |0.15 |
+ | |0.015 | ||
|- | |- | ||
|HSL | |HSL | ||
- | |49 | + | |0.49 |
+ | |0.049 | ||
|- | |- | ||
|pCinHSL | |pCinHSL | ||
- | | | + | |0.07 |
+ | |0.007 | ||
|- | |- | ||
|pCinHSLRBS | |pCinHSLRBS | ||
- | |12 | + | |0.12 |
+ | |0.012 | ||
|- | |- | ||
|RBS | |RBS | ||
- | |14 | + | |0.14 |
+ | |0.014 | ||
|- | |- | ||
|D-term | |D-term | ||
- | |27 | + | |0.27 |
+ | |0.027 | ||
|- | |- | ||
|} | |} | ||
+ | * p.s.1: the sample is diluted to 200X, and one OD is 50 ug/mL | ||
+ | * p.s.2: the calculation of concentration should be OD multiplied only by 10, not by 1,000 (It is a mistake) | ||
+ | * p.s.3: 50 ug/mL = 0.05 ug/uL, and sample is diluted to 200X. Thus the original concentration should be 0.05 ug/uL * 200 = 10 ug/uL for per OD | ||
+ | * p.s.4: [http://www.eppendorfna.com/products/quant_biophotometer_de.asp Eppendorf®Biophotometer] | ||
+ | ---- | ||
+ | |||
+ | ==Phase 1 assembly== | ||
+ | * '''Objective: pOmpC + (RBS+EYFP)''' | ||
+ | *Ligation | ||
+ | **Insert:vector=3:1 | ||
+ | **Vector: 300ng/ul(2ul pOmpC after enzyme digestion) | ||
+ | **Insert: 100ng/ul(2ul EYFP after enzyme digestion) | ||
+ | **Ligase: 1ul | ||
+ | **Final volume: 10ul | ||
+ | **RT 3h | ||
+ | *Enzyme Digestion (pOmpC) | ||
+ | **pOmpC: 14ul | ||
+ | **SpeI: 1ul | ||
+ | **PstI: 1ul | ||
+ | **10x buffer: 2, 2ul | ||
+ | **10x BSA: 2ul | ||
+ | **Total: 20ul | ||
+ | **37°C, 2h | ||
+ | **Heat inactivation: 65°C 20min | ||
+ | *Enzyme Digestion (EYFP) | ||
+ | **EYFP: 3ul | ||
+ | **XbaI: 1ul | ||
+ | **PstI: 1ul | ||
+ | **10x buffer: 3, 2ul | ||
+ | **10x BSA: 2ul | ||
+ | **H2O: 11ul | ||
+ | **Total: 20ul | ||
+ | **37°C, 2h | ||
+ | **Heat inactivation: 65°C 20min | ||
+ | ---- | ||
+ | |||
+ | ==Others== | ||
+ | *liquid culture for tar-EnvZ, pLacIQ in 5 ml LB (Kan:20ug/ml) | ||
+ | ---- | ||
+ | |||
+ | ==Who attend?== | ||
+ | * 立翔 | ||
+ | * 德銘 | ||
+ | * 惇茹 | ||
+ | * 香君 | ||
+ | * 宥嫺 |
Latest revision as of 06:19, 16 September 2007
Contents |
plasmid concentration after extraction
part | concentration (ug/ul) | A260 (OD) |
---|---|---|
pOmpC | 0.06 | 0.006 |
EYFP | 0.3 | 0.030 |
ECFP | 0.23 | 0.023 |
CinR | 0.15 | 0.015 |
HSL | 0.49 | 0.049 |
pCinHSL | 0.07 | 0.007 |
pCinHSLRBS | 0.12 | 0.012 |
RBS | 0.14 | 0.014 |
D-term | 0.27 | 0.027 |
- p.s.1: the sample is diluted to 200X, and one OD is 50 ug/mL
- p.s.2: the calculation of concentration should be OD multiplied only by 10, not by 1,000 (It is a mistake)
- p.s.3: 50 ug/mL = 0.05 ug/uL, and sample is diluted to 200X. Thus the original concentration should be 0.05 ug/uL * 200 = 10 ug/uL for per OD
- p.s.4: [http://www.eppendorfna.com/products/quant_biophotometer_de.asp Eppendorf®Biophotometer]
Phase 1 assembly
- Objective: pOmpC + (RBS+EYFP)
- Ligation
- Insert:vector=3:1
- Vector: 300ng/ul(2ul pOmpC after enzyme digestion)
- Insert: 100ng/ul(2ul EYFP after enzyme digestion)
- Ligase: 1ul
- Final volume: 10ul
- RT 3h
- Enzyme Digestion (pOmpC)
- pOmpC: 14ul
- SpeI: 1ul
- PstI: 1ul
- 10x buffer: 2, 2ul
- 10x BSA: 2ul
- Total: 20ul
- 37°C, 2h
- Heat inactivation: 65°C 20min
- Enzyme Digestion (EYFP)
- EYFP: 3ul
- XbaI: 1ul
- PstI: 1ul
- 10x buffer: 3, 2ul
- 10x BSA: 2ul
- H2O: 11ul
- Total: 20ul
- 37°C, 2h
- Heat inactivation: 65°C 20min
Others
- liquid culture for tar-EnvZ, pLacIQ in 5 ml LB (Kan:20ug/ml)
Who attend?
- 立翔
- 德銘
- 惇茹
- 香君
- 宥嫺