Team:Stockholm/20 September 2010

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==Andreas==
==Andreas==
 +
===Assembly of new parts===
 +
 +
#'''pSB1K3.N-LMWP⋅SOD⋅His'''
 +
#*Dig pSB1C3.N-LMWP (E+A)
 +
#*Dig pMA.SOD⋅His (N+P)
 +
#*Dig pSB1K3.RFP (E+P)
 +
#'''pSB1C3.N-LMWP⋅SOD⋅His'''
 +
#*Dig pSB1C3.N-LMWP (A+S)
 +
#*Dig pMA.SOD⋅His (N+S)
 +
 +
====Digestions====
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
| 
 +
!width="50"|pSB1C3. N-LMWP
 +
!width="50"|pMA. SOD⋅His
 +
!width="50"|pSB1C3. N-LMWP
 +
!width="50"|pSB1K3. N-TAT⋅SOD⋅ His 4
 +
|-
 +
|10X FastDigest buffer
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|3
 +
|align="center"|2
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|15.2
 +
|align="center"|4.1
 +
|align="center"|15.2
 +
|align="center"|11.4
 +
|-
 +
|DNA (1 &mu;g)
 +
|align="center"|9.8
 +
|align="center"|20.9
 +
|align="center"|9.8
 +
|align="center"|4.6
 +
|-
 +
|AgeI
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|NgoMIV
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|0
 +
|-
 +
|FD SpeI
 +
|align="center"|1
 +
|align="center"|1
 +
|align="center"|0
 +
|align="center"|0
 +
|-
 +
|FD EcoRI
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|align="center"|0
 +
|-
 +
|FD PstI
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|-
 +
|FD XbaI
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|0
 +
|align="center"|1
 +
|-
 +
|&nbsp;
 +
!30 &mu;l
 +
!30 &mu;l
 +
!30 &mu;l
 +
!20 &mu;l
 +
|}
 +
*Incubation: 37 &deg;C, 2:00 (NgoMIV & AgeI); 0:30 (FD)
 +
*Inactivation: 80 &deg;C, 20 min
 +
 +
====Gel verification====
 +
1.5 % agarose, 120 V
 +
 +
'''Expected bands'''
 +
*Dig pSB1C3.N-LMWP A+S 20/9: 2118 bp, (14 bp)
 +
*Dig pMA.SOD&sdot;His N+S 20/9: 2416 bp, 503 bp
 +
*Dig pSB1C3.N-LMWP E+A 20/9: 2063 bp, 69 bp
 +
*Dig pSB1K3.N-TAT&sdot;SOD&sdot;His 4 X+P 20/9: &asymp;2200 bp, 558 bp
 +
 +
'''Results'''<br />
 +
 +
====Ligations====
 +
*[Dig pSB1K3.RFP E+P 14/9] = 66.6 ng/&mu;l
 +
*[Dig pMA.His&sdot;SOD E+A 14/9] = 66.6 ng/&mu;l
 +
*[Dig pSB1C3.C-TAT N+P 15/9] = 66.6 ng/&mu;l
 +
*[Dig pSB1C3.N-LMWP A+S 20/9] = 33.3 ng/&mu;l
 +
*[Dig pMA.SOD&sdot;His N+S 20/9] = 33.3 ng/&mu;l
 +
*[Dig pSB1C3.N-LMWP E+A 20/9] = 33.3 ng/&mu;l
 +
*[Dig pSB1K3.N-TAT&sdot;SOD&sdot;His 4 X+P 20/9] = 33.3 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|&nbsp;
 +
!width="80"|pSB1C3. N-LMWP&sdot;SH
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!width="80"|pSB1K3. N-LMWP&sdot;SH
 +
!width="80"|pSB1A2. RBS.yCCS
 +
!width="80"|pEX. N-TAT&sdot;SH
 +
|-
 +
|10X T4 Ligase buffer
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|align="center"|2
 +
|-
 +
|dH<sub>2</sub>O
 +
|align="center"|9
 +
|align="center"|0
 +
|align="center"|11
 +
|align="center"|11
 +
|-
 +
|Vector DNA
 +
|align="center"|2
 +
|align="center"|1.5
 +
|align="center"|1.5
 +
|align="center"|1.5
 +
|-
 +
|Insert 1 DNA
 +
|align="center"|6
 +
|align="center"|4.5
 +
|align="center"|4.5
 +
|align="center"|4.5
 +
|-
 +
|Insert 2 DNA
 +
|align="center"|&ndash;
 +
|align="center"|11
 +
|align="center"|&ndash;
 +
|align="center"|&ndash;
 +
|-
 +
|T4 DNA ligase
 +
|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
 +
|}
 +
*Incubation: 22 &deg;C, 15 min
 +
 +
====Transformations====
 +
Standard transformations, procedures according to protocol.
 +
*1 &mu;l ligation mix
 +
**Lig pSB1C3.N-LMWP&sdot;SH (Cm 25)
 +
**Lig pSB1K3.N-LMWP&sdot;SH (Km 50)
 +
**Lig pSB1A2.RBS.yCCS (Amp 100)
 +
**Lig pEX.N-TAT&sdot;SH (Amp 100 + 50 &mu;l 0.1 mM IPTG)
 +
 +
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== his.SOD.cTAT ===
 +
 +
==== Gel ====
 +
 +
 +
{|
 +
! well
 +
! sample
 +
|-
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| pSB1C3.his.SOD.cTAT 1
 +
|-
 +
| 3
 +
| pSB1C3.his.SOD.cTAT 2
 +
|-
 +
| 4
 +
| pSB1C3.his.SOD.cTAT 3
 +
|-
 +
| 5
 +
| pSB1C3.his.SOD.cTAT 4
 +
|-
 +
| 6
 +
| pSB1C3.his.SOD.cTAT 5
 +
|-
 +
| 7
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| pSB1C3.his.SOD.cTAT 6
 +
|-
 +
| 8
 +
| pSB1C3.his.SOD
 +
|-
 +
| 9
 +
| pEX.SOD
 +
|}
 +
 +
 +
 +
 +
==== PhastGel ====
 +
 +
{|
 +
! well
 +
! sample
 +
| rowspan="9" | [[Image:place_for_picture.jpg|200px|thumb|left|]]
 +
|-
 +
| 1
 +
| SOD.his 0h
 +
|-
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| 2
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| SOD.his 2h 1:1.5
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|-
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| 3
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| his.SOD 0h
 +
|-
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| 4
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| his.SOD 2h 1:2
 +
|-
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| 5
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| yCCS 1 2h 1:2
 +
|-
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| 6
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| ladder
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|}
 +
 +
 +
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=== pEX.SOD.his ===
 +
 +
==== Gel ====
 +
 +
[[Image:2010-09-20_pEX.SOD.jpg|200px|thumb|left|]]
 +
{|
 +
! well
 +
! sample
 +
|-
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| pEX.SOD.his
 +
|-
 +
| 3
 +
| pEX.his.SOD
 +
|-
 +
| 4
 +
| yCCS 1
 +
|-
 +
| 5
 +
| yCCS 2
 +
|}
 +
 +
*Very weak bands, and in the wrong size...
 +
 +
==Johan==
 +
 +
* Cut tyrosinase with NgoMIV & SpeI
 +
 +
10 µl DNA
 +
 +
2 µl 10x buffer
 +
 +
1 µl NgoMIV
 +
 +
1 µl SpeI
 +
 +
6 µl H2O
 +
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* Cut bFGF with BamHI
 +
 +
2 µl DNA
 +
 +
2 µl 10x buffer
 +
 +
(1 µl BamHI)
 +
 +
15 µl H2O
 +
 +
Did a gel and showed that the bFGF are correct
 +
 +
* Ligated tyrosinase into pMA (vector with histag)
 +
 +
1 µl pMA
 +
 +
2 µl tyrosinase
 +
 +
2 µl 10x buffer
 +
 +
1 µl T4 ligase
 +
 +
14 µl H2O
 +
 +
{{Stockholm/Footer}}

Latest revision as of 01:34, 28 October 2010

Home Project Idea Lab Work Results Modelling Team       Follow us on and        2010.igem.org

↳   Notebook Safety Protocols


Contents

Andreas

Assembly of new parts

  1. pSB1K3.N-LMWP⋅SOD⋅His
    • Dig pSB1C3.N-LMWP (E+A)
    • Dig pMA.SOD⋅His (N+P)
    • Dig pSB1K3.RFP (E+P)
  2. pSB1C3.N-LMWP⋅SOD⋅His
    • Dig pSB1C3.N-LMWP (A+S)
    • Dig pMA.SOD⋅His (N+S)

Digestions

  pSB1C3. N-LMWP pMA. SOD⋅His pSB1C3. N-LMWP pSB1K3. N-TAT⋅SOD⋅ His 4
10X FastDigest buffer 3 3 3 2
dH2O 15.2 4.1 15.2 11.4
DNA (1 μg) 9.8 20.9 9.8 4.6
AgeI 1 0 1 0
NgoMIV 0 1 0 0
FD SpeI 1 1 0 0
FD EcoRI 0 0 1 0
FD PstI 0 0 0 1
FD XbaI 0 0 0 1
  30 μl 30 μl 30 μl 20 μl
  • Incubation: 37 °C, 2:00 (NgoMIV & AgeI); 0:30 (FD)
  • Inactivation: 80 °C, 20 min

Gel verification

1.5 % agarose, 120 V

Expected bands

  • Dig pSB1C3.N-LMWP A+S 20/9: 2118 bp, (14 bp)
  • Dig pMA.SOD⋅His N+S 20/9: 2416 bp, 503 bp
  • Dig pSB1C3.N-LMWP E+A 20/9: 2063 bp, 69 bp
  • Dig pSB1K3.N-TAT⋅SOD⋅His 4 X+P 20/9: ≈2200 bp, 558 bp

Results

Ligations

  • [Dig pSB1K3.RFP E+P 14/9] = 66.6 ng/μl
  • [Dig pMA.His⋅SOD E+A 14/9] = 66.6 ng/μl
  • [Dig pSB1C3.C-TAT N+P 15/9] = 66.6 ng/μl
  • [Dig pSB1C3.N-LMWP A+S 20/9] = 33.3 ng/μl
  • [Dig pMA.SOD⋅His N+S 20/9] = 33.3 ng/μl
  • [Dig pSB1C3.N-LMWP E+A 20/9] = 33.3 ng/μl
  • [Dig pSB1K3.N-TAT⋅SOD⋅His 4 X+P 20/9] = 33.3 ng/μl
  pSB1C3. N-LMWP⋅SH pSB1K3. N-LMWP⋅SH pSB1A2. RBS.yCCS pEX. N-TAT⋅SH
10X T4 Ligase buffer 2 2 2 2
dH2O 9 0 11 11
Vector DNA 2 1.5 1.5 1.5
Insert 1 DNA 6 4.5 4.5 4.5
Insert 2 DNA – 11 – –
T4 DNA ligase 1 1 1 1
  20 μl 20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformations

Standard transformations, procedures according to protocol.

  • 1 μl ligation mix
    • Lig pSB1C3.N-LMWP⋅SH (Cm 25)
    • Lig pSB1K3.N-LMWP⋅SH (Km 50)
    • Lig pSB1A2.RBS.yCCS (Amp 100)
    • Lig pEX.N-TAT⋅SH (Amp 100 + 50 μl 0.1 mM IPTG)




Mimmi

his.SOD.cTAT

Gel

well sample
1 ladder
2 pSB1C3.his.SOD.cTAT 1
3 pSB1C3.his.SOD.cTAT 2
4 pSB1C3.his.SOD.cTAT 3
5 pSB1C3.his.SOD.cTAT 4
6 pSB1C3.his.SOD.cTAT 5
7 pSB1C3.his.SOD.cTAT 6
8 pSB1C3.his.SOD
9 pEX.SOD



PhastGel

well sample
Place for picture.jpg
1 SOD.his 0h
2 SOD.his 2h 1:1.5
3 his.SOD 0h
4 his.SOD 2h 1:2
5 yCCS 1 2h 1:2
6 ladder


pEX.SOD.his

Gel

2010-09-20 pEX.SOD.jpg
well sample
1 ladder
2 pEX.SOD.his
3 pEX.his.SOD
4 yCCS 1
5 yCCS 2
  • Very weak bands, and in the wrong size...

Johan

  • Cut tyrosinase with NgoMIV & SpeI

10 µl DNA

2 µl 10x buffer

1 µl NgoMIV

1 µl SpeI

6 µl H2O

  • Cut bFGF with BamHI

2 µl DNA

2 µl 10x buffer

(1 µl BamHI)

15 µl H2O

Did a gel and showed that the bFGF are correct

  • Ligated tyrosinase into pMA (vector with histag)

1 µl pMA

2 µl tyrosinase

2 µl 10x buffer

1 µl T4 ligase

14 µl H2O





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

Retrieved from "http://2010.igem.org/Team:Stockholm/20_September_2010"