Team:Stockholm/9 September 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
 +
 +
===Cloning of N-CPPs into pSB1C3===
 +
Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.
 +
 +
====Digestion of N-CPP cluster====
 +
[N-CPP plasmid] = 672 ng/μl
 +
 +
''Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.''
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
| 
 +
!width="50"|N-CPP
 +
|-
 +
|colspan="2" align="center"|1st incubation
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|3
 +
|-
 +
|DNA (2 μg)
 +
|align="center"|3
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|19
 +
|-
 +
|XbaI (conv.)
 +
|align="center"|1
 +
|-
 +
|AgeI (conv.)
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!27 &mu;l
 +
|-
 +
|colspan="2" align="center"|2nd incubation
 +
|-
 +
|FD BamHI
 +
|align="center"|1
 +
|-
 +
|FD HindIII
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!29 &mu;l
 +
|}
 +
 +
*'''1st incubation:''' 37 &deg;C, 2:30
 +
*'''2nd incubation:''' 37 &deg;C, 0:30
 +
*'''Inactivation:''' 80 &deg;C, 20 min
 +
 +
====Ligation====
 +
Two ligation reactions were prepared to test the efficiency of two different ligation buffers.
 +
 +
*'''Vector:''' Dig pSB1C3 X+A EXTR (13.72 ng/&mu;l)
 +
*'''Insert:''' Dig N-CPP X+A 9/9 (31.25 ng/&mu;l)
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!Lig pSB1C3<br />NCPP 1<br />9/9
 +
!Lig pSB1C3<br />NCPP 2<br />9/9
 +
|-
 +
|5X Rapid Ligation buf.
 +
|align="center"|4
 +
|align="center"|0
 +
|-
 +
|10X T4 DNA ligase buf.
 +
|align="center"|0
 +
|align="center"|2
 +
|-
 +
|Vector DNA
 +
|align="center"|4
 +
|align="center"|4
 +
|-
 +
|Insert DNA
 +
|align="center"|11
 +
|align="center"|11
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|0
 +
|align="center"|2
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
 +
*Incubation: 22 &deg;C, 16 min
 +
 +
====Digestion of previous ligation sample====
 +
 +
:'''Ligation mix:''' Lig pSB1C3.N-CPP* 6/9
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|Ligation mix
 +
|align="center"|15
 +
|-
 +
|10X FD buffer
 +
|align="center"|2
 +
|-
 +
|FD BamHI
 +
|align="center"|1
 +
|-
 +
|FD HindIII
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!19 &mu;l
 +
|}
 +
 +
*Incubation: 37 &deg;C, 30 min
 +
*Inactivation: 80 &deg;C, 15 min
 +
 +
====Transformations====
 +
Standard transformation protocol.
 +
*3 &mu;l ligation mix
 +
**Lig pSB1C3.N-CPP 1 9/9
 +
**Lig pSB1C3.N-CPP 2 9/9
 +
**Lig pSB1C3.N-CPP * 6/9
 +
*Cm 25 plates
 +
 +
===Joint expression of SOD and yCCS from pEX===
 +
 +
Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by [http://www.ncbi.nlm.nih.gov/pubmed/15358352 Ahl, Lindberg and Tibell (2004)], we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.<br />
 +
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.

Revision as of 08:40, 13 September 2010

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Contents

Andreas

Cloning of N-CPPs into pSB1C3

Since we realized that the method we used for cloning the N-CPPs can cause also the intervening sequences to insert into pSB1C3, I decided to redo some clonings. Since the intervening sequences were designed with unique restriction sites, digestion with these endonucleases should prevent cloning of these.

Digestion of N-CPP cluster

[N-CPP plasmid] = 672 ng/μl

Tested the FastDigest buffer, even though conventional Fermentas restriction enzymes were used.

  N-CPP
1st incubation
10X FastDigest buffer 3
DNA (2 μg) 3
dH2O 19
XbaI (conv.) 1
AgeI (conv.) 1
  27 μl
2nd incubation
FD BamHI 1
FD HindIII 1
  29 μl
  • 1st incubation: 37 °C, 2:30
  • 2nd incubation: 37 °C, 0:30
  • Inactivation: 80 °C, 20 min

Ligation

Two ligation reactions were prepared to test the efficiency of two different ligation buffers.

  • Vector: Dig pSB1C3 X+A EXTR (13.72 ng/μl)
  • Insert: Dig N-CPP X+A 9/9 (31.25 ng/μl)
  Lig pSB1C3
NCPP 1
9/9 		Lig pSB1C3
NCPP 2
9/9
5X Rapid Ligation buf. 4 0
10X T4 DNA ligase buf. 0 2
Vector DNA 4 4
Insert DNA 11 11
dH2O 0 2
T4 DNA ligase 1 1
  20 μl 20 μl
  • Incubation: 22 °C, 16 min

Digestion of previous ligation sample

Ligation mix: Lig pSB1C3.N-CPP* 6/9
Ligation mix 15
10X FD buffer 2
FD BamHI 1
FD HindIII 1
  19 μl
  • Incubation: 37 °C, 30 min
  • Inactivation: 80 °C, 15 min

Transformations

Standard transformation protocol.

  • 3 μl ligation mix
    • Lig pSB1C3.N-CPP 1 9/9
    • Lig pSB1C3.N-CPP 2 9/9
    • Lig pSB1C3.N-CPP * 6/9
  • Cm 25 plates

Joint expression of SOD and yCCS from pEX

Me and Mimmi were discussing the upcoming expression of SOD and its helper chaperone yCCS. Based on an article by [http://www.ncbi.nlm.nih.gov/pubmed/15358352 Ahl, Lindberg and Tibell (2004)], we decided that the two proteins should be expressed in equal amounts (1:1) from the same vector. Since we only have one expression vector (pEX) available, this requires some modifications.
Our idea is to construct a SOD/yCCS operon from which the two genes can be co-transcribed. This will require a new Shine-Dalgarno (RBS) sequence for translation of the second gene in the operon.

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