Team:Stockholm/25 September 2010

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==Andreas==
==Andreas==
 +
 +
===Cloning and assembly===
 +
====Digestion====
 +
Further digestions in addition to the ones performed 24/9.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
| 
 +
!width="70"|pK.N-TAT⋅ SH (4) X+P
 +
!width="70"|pK.N-LMWP⋅ SH (1) X+P
 +
!width="70"|pK.N-LMWP⋅ SH (1) S+P
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|DNA (1 μg)
 +
|align="center"|8
 +
|align="center"|4
 +
|align="center"|4
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|8
 +
|align="center"|12
 +
|align="center"|12
 +
|-
 +
|FD XbaI
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|FD SpeI
 +
|align="center"|0
 +
|align="center"|0
 +
|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, 1 h
 +
*Inactivation: 80 &deg;C, 30 min (including digestion samples from 24/9)
 +
 +
====Ligation====
 +
''Underlined sequences relevant for assembly''
 +
 +
{|border="1" cellpadding="1" cellspacing="0" align="right"
 +
|&nbsp;
 +
!width="50"|1
 +
!width="50"|2
 +
!width="50"|3
 +
!width="50"|4
 +
!width="50"|5
 +
!width="50"|6
 +
!width="50"|7
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!width="50"|8
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|-
 +
|'''10X T4 Ligase buffer'''
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|'''Vector DNA (50 ng)'''
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|'''Insert DNA'''
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|align="center"|5
 +
|align="center"|5
 +
|align="center"|5
 +
|align="center"|5
 +
|align="center"|4
 +
|align="center"|4
 +
|align="center"|4
 +
|align="center"|4
 +
|-
 +
|'''dH<sub>2</sub>O'''
 +
|align="center"|11
 +
|align="center"|11
 +
|align="center"|11
 +
|align="center"|11
 +
|align="center"|12
 +
|align="center"|12
 +
|align="center"|12
 +
|align="center"|12
 +
|-
 +
|'''T4 DNA ligase'''
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
!20 &mu;l
 +
|}
 +
#'''pEX.N-LMWP&sdot;SOD&sdot;His (K)'''
 +
#*pSB1K3.<u>N-LMWP&sdot;SOD&sdot;His</u> X+P
 +
#*<u>pEX</u>.RFP X+P
 +
#'''pEX.N-TAT&sdot;SOD&sdot;His'''
 +
#*pSB1K3.<u>N-TAT&sdot;SOD&sdot;His</u> X+P
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#*<u>pEX</u>.RFP X+P
 +
#'''pEX.N-Tra10&sdot;SOD&sdot;His'''
 +
#*pSB1K3.<u>N-Tra10&sdot;SOD&sdot;His</u> X+P
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#*<u>pEX</u>.RFP X+P
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#'''pEX.N-LMWP&sdot;SOD&sdot;His (C)'''
 +
#*pSB1C3.<u>N-LMWP&sdot;SOD&sdot;His</u> X+P
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#*<u>pEX</u>.RFP X+P
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#'''pSB1K3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS'''
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#*<u>pSB1K3.N-LMWP&sdot;SOD&sdot;His</u> S+P
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#*pSB1A2<u>.RBS.yCCS</u> X+P
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#'''pSB1K3.N-TAT&sdot;SOD&sdot;His.RBS.yCCS'''
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#*<u>pSB1K3.N-TAT&sdot;SOD&sdot;His</u> S+P
 +
#*pSB1A2<u>.RBS.yCCS</u> X+P
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#'''pSB1K3.N-Tra10&sdot;SOD&sdot;His.RBS.yCCS'''
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#*<u>pSB1K3.N-Tra10&sdot;SOD&sdot;His</u> S+P
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#*pSB1A2<u>.RBS.yCCS</u> X+P
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#'''pSB1C3.N-LMWP&sdot;SOD&sdot;His.RBS.yCCS'''
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#*<u>pSB1C3.N-LMWP&sdot;SOD&sdot;His</u> S+P
 +
#*pSB1A2<u>.RBS.yCCS</u> X+P
 +
 +
====Transformation====
 +
*Standard transformation according to protocol.
 +
**1 &mu;l ligation mix
 +
**Amp 100, Cm 25 or Km 50 plates
 +
**50 &mu;l IPTG (pEX constructs, Amp 100 plates)
 +
 +
===Sequencing preparation===
 +
Prepared samples for sequencing to be sent on Monday.
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!Construct
 +
!Primer
 +
!Sample name
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!Sequencing code<br />(edited 27/9)
 +
|-
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|pEX.N-TAT&sdot;SOD&sdot;His 3
 +
|pEXf
 +
|pEX.nTAT*SH3_pEXf
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|ASB0045 614
 +
|-
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|pEX.N-TAT&sdot;SOD&sdot;His 4
 +
|pEXf
 +
|pEX.nTAT*SH4_pEXf
 +
|ASB0045 615
 +
|-
 +
|pSB1A2.RBS.yCCS 3
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|VF2
 +
|pA.RBS.yCCS3_VF2
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|ASB0045 603
 +
|-
 +
|pSB1A2.RBS.yCCS 4
 +
|VF2
 +
|pA.RBS.yCCS4_VF2
 +
|ASB0045 604
 +
|-
 +
|pSB1K3.N-TAT&sdot;SOD&sdot;His 4
 +
|VF2
 +
|pK.nTAT*SH4_VF2
 +
|ASB0045 608
 +
|-
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|pSB1K3.N-TAT&sdot;SOD&sdot;His 5
 +
|VF2
 +
|pK.nTAT*SH5_VF2
 +
|ASB0045 609
 +
|-
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|pSB1K3.N-Tra10&sdot;SOD&sdot;His 5
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|VF2
 +
|pK.nTra10*SH5_VF2
 +
|ASB0045 610
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|-
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|pSB1K3.N-LMWP&sdot;SOD&sdot;His 1
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|VF2
 +
|pK.nLMWP*SH1_VF2
 +
|ASB0045 607
 +
|-
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|pSB1C3.N-LMWP&sdot;SOD&sdot;His 1
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|VF2
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|pC.nLMWP*SH1_VF2
 +
|ASB0045 605
 +
|-
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|pSB1C3.N-LMWP&sdot;SOD&sdot;His 4
 +
|VF2
 +
|pC.nLMWP*SH4_VF2
 +
|ASB0045 606
 +
|-
 +
|pEX.SOD
 +
|pEXf
 +
|pEX.SOD_pEXf
 +
|ASB0045 611
 +
|-
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|pEX.yCCS 5
 +
|pEXf
 +
|pEX.yCCS5_pEXf
 +
|ASB0045 616
 +
|-
 +
|pEX.SOD&sdot;His
 +
|pEXf
 +
|pEX.SH_pEXf
 +
|ASB0045 612
 +
|-
 +
|pEX.His&sdot;SOD
 +
|pEXf
 +
|pEX.HS_pEXf
 +
|ASB0045 613
 +
|}
 +
 +
===BL21 transformation===
 +
Transformed BL21 cells with not-yet-verified pEX.N-TAT&sdot;SOD&sdot;His plasmid samples 3 & 4
 +
*Standard transformation according to protocol.
 +
**1 &mu;l ligation mix
 +
***100 &mu;l cell aliquot divided in 2x50 &mu;l to lower number of colonies after transformation.
 +
**Amp 100 plates
 +
 +
===Preparation of LB agar plates===
 +
Made new Cm (25 &mu;g/ml) and Km (50 &mu;g/ml) LB agar plates.
 +
*10X Cm 25
 +
*10X Km 50
 +
 +
{{Stockholm/Footer}}

Latest revision as of 11:06, 26 October 2010

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Contents

Andreas

Cloning and assembly

Digestion

Further digestions in addition to the ones performed 24/9.

  pK.N-TAT⋅ SH (4) X+P pK.N-LMWP⋅ SH (1) X+P pK.N-LMWP⋅ SH (1) S+P
10X FastDigest buffer 2 2 2
DNA (1 μg) 8 4 4
dH2O 8 12 12
FD XbaI 1 1 0
FD SpeI 0 0 1
FD PstI 1 1 1
  20 μl 20 μl 20 μl
  • Incubation: 37 °C, 1 h
  • Inactivation: 80 °C, 30 min (including digestion samples from 24/9)

Ligation

Underlined sequences relevant for assembly

  1 2 3 4 5 6 7 8
10X T4 Ligase buffer 2 2 2 2 2 2 2 2
Vector DNA (50 ng) 1 1 1 1 1 1 1 1
Insert DNA 5 5 5 5 4 4 4 4
dH2O 11 11 11 11 12 12 12 12
T4 DNA ligase 1 1 1 1 1 1 1 1
  20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 20 μl 20 μl
  1. pEX.N-LMWP⋅SOD⋅His (K)
    • pSB1K3.N-LMWP⋅SOD⋅His X+P
    • pEX.RFP X+P
  2. pEX.N-TAT⋅SOD⋅His
    • pSB1K3.N-TAT⋅SOD⋅His X+P
    • pEX.RFP X+P
  3. pEX.N-Tra10⋅SOD⋅His
    • pSB1K3.N-Tra10⋅SOD⋅His X+P
    • pEX.RFP X+P
  4. pEX.N-LMWP⋅SOD⋅His (C)
    • pSB1C3.N-LMWP⋅SOD⋅His X+P
    • pEX.RFP X+P
  5. pSB1K3.N-LMWP⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-LMWP⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  6. pSB1K3.N-TAT⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-TAT⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  7. pSB1K3.N-Tra10⋅SOD⋅His.RBS.yCCS
    • pSB1K3.N-Tra10⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P
  8. pSB1C3.N-LMWP⋅SOD⋅His.RBS.yCCS
    • pSB1C3.N-LMWP⋅SOD⋅His S+P
    • pSB1A2.RBS.yCCS X+P

Transformation

  • Standard transformation according to protocol.
    • 1 μl ligation mix
    • Amp 100, Cm 25 or Km 50 plates
    • 50 μl IPTG (pEX constructs, Amp 100 plates)

Sequencing preparation

Prepared samples for sequencing to be sent on Monday.

Construct Primer Sample name Sequencing code
(edited 27/9)
pEX.N-TAT⋅SOD⋅His 3 pEXf pEX.nTAT*SH3_pEXf ASB0045 614
pEX.N-TAT⋅SOD⋅His 4 pEXf pEX.nTAT*SH4_pEXf ASB0045 615
pSB1A2.RBS.yCCS 3 VF2 pA.RBS.yCCS3_VF2 ASB0045 603
pSB1A2.RBS.yCCS 4 VF2 pA.RBS.yCCS4_VF2 ASB0045 604
pSB1K3.N-TAT⋅SOD⋅His 4 VF2 pK.nTAT*SH4_VF2 ASB0045 608
pSB1K3.N-TAT⋅SOD⋅His 5 VF2 pK.nTAT*SH5_VF2 ASB0045 609
pSB1K3.N-Tra10⋅SOD⋅His 5 VF2 pK.nTra10*SH5_VF2 ASB0045 610
pSB1K3.N-LMWP⋅SOD⋅His 1 VF2 pK.nLMWP*SH1_VF2 ASB0045 607
pSB1C3.N-LMWP⋅SOD⋅His 1 VF2 pC.nLMWP*SH1_VF2 ASB0045 605
pSB1C3.N-LMWP⋅SOD⋅His 4 VF2 pC.nLMWP*SH4_VF2 ASB0045 606
pEX.SOD pEXf pEX.SOD_pEXf ASB0045 611
pEX.yCCS 5 pEXf pEX.yCCS5_pEXf ASB0045 616
pEX.SOD⋅His pEXf pEX.SH_pEXf ASB0045 612
pEX.His⋅SOD pEXf pEX.HS_pEXf ASB0045 613

BL21 transformation

Transformed BL21 cells with not-yet-verified pEX.N-TAT⋅SOD⋅His plasmid samples 3 & 4

  • Standard transformation according to protocol.
    • 1 μl ligation mix
      • 100 μl cell aliquot divided in 2x50 μl to lower number of colonies after transformation.
    • Amp 100 plates

Preparation of LB agar plates

Made new Cm (25 μg/ml) and Km (50 μg/ml) LB agar plates.

  • 10X Cm 25
  • 10X Km 50





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/

Retrieved from "http://2010.igem.org/Team:Stockholm/25_September_2010"