Team:Stockholm/1 September 2010

From 2010.igem.org

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(New page: {{Stockholm/Top2}} ==Andreas==)
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==Andreas==
==Andreas==
 +
 +
===His⋅SOD cloning into pMA===
 +
 +
====Digestion====
 +
 +
[pSB1C3.m-SOD]=105.5 ng/&mu;l<br />
 +
[pMA.His]=85.67 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|align="center"|[&mu;l]
 +
!width="50"|SOD
 +
!width="50"|pMA.His
 +
|-
 +
|10X FD buffer
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|DNA (2 &mu;g)
 +
|align="center"|19
 +
|align="center"|23
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|6
 +
|align="center"|3
 +
|-
 +
|FD AgeI
 +
|align="center"|0
 +
|align="center"|0.5
 +
|-
 +
|NgoMIV
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|FD SpeI
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!30
 +
!30.5
 +
|}
 +
 +
'''Incubation:'''
 +
*37 &deg;C, 0:30 (FD)
 +
*37 &deg;C, 2:00 (NgoMIV)
 +
 +
'''Inactivation:'''
 +
*80 &deg;C, 10 min
 +
 +
====Ligation====
 +
 +
{|border="1" cellpadding="1" cellspacing="1"
 +
|&nbsp;
 +
|align="center"|[&mu;l]
 +
|-
 +
|5X Rapid ligation buf.
 +
|width="40" align="center"|4
 +
|-
 +
|Vector DNA (pMa.His)
 +
|align="center"|1.5
 +
|-
 +
|Insert DNA (m-SOD)
 +
|align="center"|4.5
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|9
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20
 +
|}
 +
 +
'''Incubation:'''
 +
*22 &deg;C, 10 min
 +
 +
====Transformation====
 +
Quick-transformation according to protocol into both Top10 and DH5&alpha; to compare and verify Top10 competence:
 +
*1.5 &mu;l
 +
*Amp 100
 +
 +
===SOD&sdot;His cloning into pMA===
 +
 +
====Glycerol stocks====
 +
''From 31/8 ON cultures''
 +
 +
*SH1: pMA.SOD&sdot;His 1, 1/9/2010
 +
*SH2: pMA.SOD&sdot;His 2, 1/9/2010
 +
 +
===Transfer of m-yCCS into pEX===
 +
 +
====Digestion====
 +
[pEX.RFP] = 82 ng/&mu;l
 +
[pSB1C3.m-yCCS] = 101.2 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|align="center"|[&mu;l]
 +
!width="50"|SOD
 +
!width="50"|pMA.His
 +
|-
 +
|10X FD buffer
 +
|align="center"|3
 +
|align="center"|3
 +
|-
 +
|DNA (1 &mu;g)
 +
|align="center"|12
 +
|align="center"|6
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|13
 +
|align="center"|19
 +
|-
 +
|FD XbaI
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|FD PstI
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!30
 +
!30
 +
|}
 +
 +
'''Incubation:'''
 +
*37 &deg;C, 10 min
 +
 +
'''Inactivation:'''
 +
*65 &deg;C, 20 min
 +
 +
====Ligation====
 +
 +
{|border="1" cellpadding="1" cellspacing="1"
 +
|&nbsp;
 +
|align="center"|[&mu;l]
 +
|-
 +
|5X Rapid ligation buf.
 +
|width="40" align="center"|4
 +
|-
 +
|Vector DNA (pEX)
 +
|align="center"|1.5
 +
|-
 +
|Insert DNA (m-yCCS)
 +
|align="center"|9
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|4.5
 +
|-
 +
|T4 DNA ligase
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20
 +
|}
 +
 +
'''Incubation:'''
 +
*22 &deg;C, 10 min
 +
 +
====Transformation====
 +
Quick-transformation (Top10)
 +
*2 &mu;l
 +
*Amp 100

Revision as of 21:57, 3 September 2010

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Contents

Andreas

His⋅SOD cloning into pMA

Digestion

[pSB1C3.m-SOD]=105.5 ng/μl
[pMA.His]=85.67 ng/μl

[μl] SOD pMA.His
10X FD buffer 3 3
DNA (2 μg) 19 23
dH2O 6 3
FD AgeI 0 0.5
NgoMIV 1 0
FD SpeI 1 1
  30 30.5

Incubation:

  • 37 °C, 0:30 (FD)
  • 37 °C, 2:00 (NgoMIV)

Inactivation:

  • 80 °C, 10 min

Ligation

  [μl]
5X Rapid ligation buf. 4
Vector DNA (pMa.His) 1.5
Insert DNA (m-SOD) 4.5
dH2O 9
T4 DNA ligase 1
  20

Incubation:

  • 22 °C, 10 min

Transformation

Quick-transformation according to protocol into both Top10 and DH5α to compare and verify Top10 competence:

  • 1.5 μl
  • Amp 100

SOD⋅His cloning into pMA

Glycerol stocks

From 31/8 ON cultures

  • SH1: pMA.SOD⋅His 1, 1/9/2010
  • SH2: pMA.SOD⋅His 2, 1/9/2010

Transfer of m-yCCS into pEX

Digestion

[pEX.RFP] = 82 ng/μl [pSB1C3.m-yCCS] = 101.2 ng/μl

[μl] SOD pMA.His
10X FD buffer 3 3
DNA (1 μg) 12 6
dH2O 13 19
FD XbaI 1 1
FD PstI 1 1
  30 30

Incubation:

  • 37 °C, 10 min

Inactivation:

  • 65 °C, 20 min

Ligation

  [μl]
5X Rapid ligation buf. 4
Vector DNA (pEX) 1.5
Insert DNA (m-yCCS) 9
dH2O 4.5
T4 DNA ligase 1
  20

Incubation:

  • 22 °C, 10 min

Transformation

Quick-transformation (Top10)

  • 2 μl
  • Amp 100
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