Team:Stockholm/16 October 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
 +
 +
===Transfer of operon constructs into pEX===
 +
''Continued Mimmi's experiments'''
 +
 +
====Plasmid prep====
 +
*40 μl elution buffer (x2)
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|-
 +
!Sample
 +
!width="60"|Conc [ng/μl]
 +
!width="60"|A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.SOD.yCCS
 +
|align="center"|188.2
 +
|align="center"|1.94
 +
|-
 +
|pSB1C3.SOD&sdot;His.yCCS
 +
|align="center"|223.6
 +
|align="center"|1.91
 +
|-
 +
|pSB1C3.His&sdot;SOD.yCCS
 +
|align="center"|230.6
 +
|align="center"|1.93
 +
|}
 +
 +
====Digestions====
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="90"|pSB1C3.<br />SOD.yCCS
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!width="90"|pSB1C3.<br />SOD&sdot;His.yCCS
 +
!width="90"|pSB1C3.<br />His&sdot;SOD.yCCS
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|DNA (1 &mu;g)
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|align="center"|5.3
 +
|align="center"|4.5
 +
|align="center"|4.3
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|10.7
 +
|align="center"|11.5
 +
|align="center"|11.7
 +
|-
 +
|FD XbaI
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|FD PstI
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
*Incubation: 37 &deg;C, 30 min
 +
*Inactivation: 80 &deg;C, 20 min
 +
 +
====Ligations====
 +
Vector: pre-digested pEX.RFP (X+P)
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="50"|pEX.SOD.<br />yCCS
 +
!width="50"|pEX.<br />SOD&sdot;His.<br />yCCS
 +
!width="50"|pEX.<br />His&sdot;SOD.<br />yCCS
 +
|-
 +
|10X T4 Ligase buffer
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|Vector DNA
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|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|Insert DNA
 +
|align="center"|9
 +
|align="center"|9
 +
|align="center"|9
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|5
 +
|align="center"|5
 +
|align="center"|5
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
*Incubation: 22 &deg;C, 15 min
 +
 +
====Transformation====
 +
''By Mimmi''
 +
 +
===nCPP culture growth assay===
 +
Prepared new clones to repeat the experiment from 15/10 by transforming new BL21.
 +
 +
Quick transformation protocol.
 +
*50 &mu;l competent BL21 (30 &mu;l plated)
 +
*0.5 &mu;l plasmid DNA
 +
**pEX.SOD&sdot;His
 +
**pEX.nTra10&sdot;SOD&sdot;His
 +
**pEX.nTAT&sdot;SOD&sdot;His
 +
**pEX.nLMWP&sdot;SOD&sdot;His

Revision as of 13:01, 16 October 2010

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Contents

Andreas

Transfer of operon constructs into pEX

Continued Mimmi's experiments'

Plasmid prep

  • 40 μl elution buffer (x2)
DNA concentration
Sample Conc [ng/μl] A260/A280
pSB1C3.SOD.yCCS 188.2 1.94
pSB1C3.SOD⋅His.yCCS 223.6 1.91
pSB1C3.His⋅SOD.yCCS 230.6 1.93

Digestions

  pSB1C3.
SOD.yCCS 		pSB1C3.
SOD⋅His.yCCS 		pSB1C3.
His⋅SOD.yCCS
10X FastDigest buffer 2 2 2
DNA (1 μg) 5.3 4.5 4.3
dH2O 10.7 11.5 11.7
FD XbaI 1 1 1
FD PstI 1 1 1
  20 μl 20 μl 20 μl
  • Incubation: 37 °C, 30 min
  • Inactivation: 80 °C, 20 min

Ligations

Vector: pre-digested pEX.RFP (X+P)

  pEX.SOD.
yCCS 		pEX.
SOD⋅His.
yCCS 		pEX.
His⋅SOD.
yCCS
10X T4 Ligase buffer 2 2 2
Vector DNA 3 3 3
Insert DNA 9 9 9
dH2O 5 5 5
T4 DNA ligase 1 1 1
  20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformation

By Mimmi

nCPP culture growth assay

Prepared new clones to repeat the experiment from 15/10 by transforming new BL21.

Quick transformation protocol.

  • 50 μl competent BL21 (30 μl plated)
  • 0.5 μl plasmid DNA
    • pEX.SOD⋅His
    • pEX.nTra10⋅SOD⋅His
    • pEX.nTAT⋅SOD⋅His
    • pEX.nLMWP⋅SOD⋅His
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