NYMU Taipei/Lab Notes/2007 8 27

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(→plasmid concentration after extraction)
 
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* plasmid concentration after liquid culture extraction
+
== 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
-
|6
+
|0.06
 +
|0.006
|-
|-
|EYFP
|EYFP
-
|30
+
|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
-
|7
+
|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]
----
----
-
==first assembly==
+
 
 +
==Phase 1 assembly==
 +
* '''Objective: pOmpC + (RBS+EYFP)'''
*Ligation
*Ligation
**Insert:vector=3:1
**Insert:vector=3:1
Line 41: Line 57:
**Final volume: 10ul
**Final volume: 10ul
**RT 3h
**RT 3h
-
*Enzyme Digestion
+
*Enzyme Digestion (pOmpC)
-
**pOmpC: 14ul EYFP: 3ul
+
**pOmpC: 14ul
-
**RE: 1ul
+
**SpeI: 1ul
 +
**PstI: 1ul
 +
**10x buffer: 2, 2ul
 +
**10x BSA: 2ul
 +
**Total: 20ul
 +
**37°C, 2h
**Heat inactivation: 65°C 20min
**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==
==Others==
-
*Culture tar-EnvZ, pLacIQ in 5 ml LB liquid medium (Kan:20ug/ml)
+
*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
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