Team:Stockholm/3 August 2010

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==Andreas==
==Andreas==
 +
 +
===Plasmid prep.===
 +
 +
''From 2/8 ON cultures''
 +
 +
Elution volume: 70 μl.
 +
 +
{|border="1" cellpadding="2" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|-
 +
!Sample
 +
!Conc. [ng/μl]
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1A3 1
 +
|87.00
 +
|1.98
 +
|-
 +
|pSB1A3 2
 +
|120.3
 +
|1.92
 +
|-
 +
|pSB1A3 3
 +
|145.7
 +
|1.94
 +
|-
 +
|pSB1A3 4
 +
|58.72
 +
|2.01
 +
|-
 +
|pSB1C3 1
 +
|188.9
 +
|1.97
 +
|-
 +
|pSB1C3 2
 +
|101.2
 +
|2.00
 +
|-
 +
|pSB1C3 3
 +
|201.2
 +
|1.93
 +
|-
 +
|pSB1C3 4
 +
|147.7
 +
|1.92
 +
|}
 +
 +
===Cloning===
 +
 +
To remove unwanted insertion of a C nucleotide located at the PstI site in the suffix, caused by cloning in the pEX vector, yCCS, SOD and IgG protease will be transferred to a new vector by digestion with SpeI.
 +
 +
====Digestion====
 +
*IgG protease (on pSB1A3)
 +
*yCCS A (on pSB1C3)
 +
*yCCS B (on pSB1C3)
 +
*SOD (on pSB1C3)
 +
*pSB1C3 2
 +
*pSB1A3 2
 +
 +
{|border="1" cellpadding="2" cellspacing="0"
 +
!colspan="7"|Digestion tubes
 +
|-
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![&mu;l]
 +
!IgGp<br>[500]
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!SOD<br>[120.6]
 +
!yCCS A<br>[94.8]
 +
!yCCS B<br>[85.8]
 +
!pSB1A3<br>[120.3]
 +
!pSB1C3<br>[101.2]
 +
|-
 +
|'''10X FastDigest buffer'''
 +
|5
 +
|5
 +
|5
 +
|5
 +
|5
 +
|5
 +
|-
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|'''dH<sub>2</sub>O'''
 +
|39
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|26
 +
|22
 +
|20
 +
|26
 +
|23
 +
|-
 +
|'''DNA (2 &mu;g)'''
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|4
 +
|17
 +
|21
 +
|23
 +
|17
 +
|20
 +
|-
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|'''FD SpeI'''&dagger;
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|0.5
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|0.5
 +
|0.5
 +
|0.5
 +
|0.5
 +
|0.5
 +
|-
 +
|'''FD EcoRI'''
 +
|1
 +
|1
 +
|1
 +
|1
 +
|1
 +
|1
 +
|-
 +
|&nbsp;
 +
|'''50'''
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|'''50'''
 +
|'''50'''
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|'''50'''
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|'''50'''
 +
|'''50'''
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|}
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&dagger; ''Only 0.5 &mu;l of FD SpeI to save enzyme, since it is very expensive.''
 +
 +
#Gentle mixing
 +
#Incubation in 37 &deg;C, 15 min
 +
#Tubes returned to ice
 +
#Promptly proceeded to ligation, '''without enzyme inactivation or DNA purification'''.
 +
 +
====Ligation====
 +
*pSB1C3 + IgG prot.
 +
*pSB1A3 + yCCS A
 +
*pSB1A3 + yCCS B
 +
*pSB1A3 + SOD
 +
 +
{|border="1" cellpadding="2" cellspacing="0"
 +
!colspan="5"|Ligation tubes (vector:insert 1:5)
 +
|-
 +
![&mu;l]
 +
!IgG + pSB1C3
 +
!SOD + pSB1A3
 +
!yCCS A + pSB1A3
 +
!yCCS B + pSB1A3
 +
|-
 +
|'''Vector DNA [100 ng]'''
 +
|2.5
 +
|2.5
 +
|2.5
 +
|2.5
 +
|-
 +
|'''Insert DNA [500 ng]'''
 +
|12.5
 +
|12.5
 +
|12.5
 +
|12.5
 +
|-
 +
|'''5X Rapid Ligation buffer'''
 +
|4
 +
|4
 +
|4
 +
|4
 +
|-
 +
|'''T4 DNA ligase'''
 +
|1
 +
|1
 +
|1
 +
|1
 +
|-
 +
|&nbsp;
 +
|'''20'''
 +
|'''20'''
 +
|'''20'''
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|'''20'''
 +
|}
 +
 +
#Gently mixing
 +
#Incubation 22 &deg;C, 10 min
 +
#Collection by 5 sec centrifugation
 +
#Tubes returned to ice
 +
 +
====Quick transformation====
 +
 +
#100 &mu;l Top10 cells thawed
 +
#2 &mu;l ligation mixture added to cells. Cells kept on ice &asymp;5 min
 +
#Heat-shock in 42 &deg;C, 30 sec
 +
#Cells plated on LB agar with relevant antibiotics
 +
#*100 Amp
 +
#*25 Cm
 +
#Incubation ON in 37 &deg;C
 +
 +
 +
 +
 +
==Mimmi==
 +
 +
=== MITF ===
 +
 +
[[Image:PRcCMV.jpg|400px|thumb|left|the vector pRc/CMV and the insert MITF]]
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
==== - restriction enzyme ====
 +
 +
 +
*the PCR products (from 2010-08-02) are treated with the restriction enzyme AgeI
 +
**the mutated MITF should show two bands: ~1300bp and ~10bp (which you dont see)
 +
**the control (non-mutated) MITF should show three bands: ~1060bp, ~200bp and ~10bp (which you don't see)
 +
 +
 +
{|
 +
! Mix
 +
| (µl)
 +
|-
 +
| DNA
 +
| 15
 +
|-
 +
| sH<sub>2</sub>O
 +
| 2
 +
|-
 +
| 10X buffer
 +
| 2
 +
|-
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| AgeI
 +
| 1
 +
|-
 +
| align="right" | tot
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| 20
 +
|}
 +
 +
 +
*Mix and spinn down breafly
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*Incubate 2h (1-16h)
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 +
 +
 +
==== - Gel ====
 +
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*0.5% agarose gel
 +
*(Melted in our bath, redoing the gel in another bath...
 +
 +
----
 +
==Nina==
 +
 +
===Overnight culture of Tyrosinase===
 +
 +
 +
I inoculated Tyrosinase in its original vector from a glycerol stock (that was sent to us from iGEM hq) in 12 ml LB and added 24 ul amphicillin (50 mg/ml). This was incubated in 37 °C overnight in shake. This will be mini prepped tomorrow.
 +
 +
{{Stockholm/Footer}}

Latest revision as of 10:46, 26 October 2010

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Contents

Andreas

Plasmid prep.

From 2/8 ON cultures

Elution volume: 70 μl.

DNA concentration
Sample Conc. [ng/μl] A260/A280
pSB1A3 1 87.00 1.98
pSB1A3 2 120.3 1.92
pSB1A3 3 145.7 1.94
pSB1A3 4 58.72 2.01
pSB1C3 1 188.9 1.97
pSB1C3 2 101.2 2.00
pSB1C3 3 201.2 1.93
pSB1C3 4 147.7 1.92

Cloning

To remove unwanted insertion of a C nucleotide located at the PstI site in the suffix, caused by cloning in the pEX vector, yCCS, SOD and IgG protease will be transferred to a new vector by digestion with SpeI.

Digestion

  • IgG protease (on pSB1A3)
  • yCCS A (on pSB1C3)
  • yCCS B (on pSB1C3)
  • SOD (on pSB1C3)
  • pSB1C3 2
  • pSB1A3 2
Digestion tubes
[μl] IgGp
[500] 		SOD
[120.6] 		yCCS A
[94.8] 		yCCS B
[85.8] 		pSB1A3
[120.3] 		pSB1C3
[101.2]
10X FastDigest buffer 5 5 5 5 5 5
dH2O 39 26 22 20 26 23
DNA (2 μg) 4 17 21 23 17 20
FD SpeI† 0.5 0.5 0.5 0.5 0.5 0.5
FD EcoRI 1 1 1 1 1 1
  50 50 50 50 50 50

† Only 0.5 μl of FD SpeI to save enzyme, since it is very expensive.

  1. Gentle mixing
  2. Incubation in 37 °C, 15 min
  3. Tubes returned to ice
  4. Promptly proceeded to ligation, without enzyme inactivation or DNA purification.

Ligation

  • pSB1C3 + IgG prot.
  • pSB1A3 + yCCS A
  • pSB1A3 + yCCS B
  • pSB1A3 + SOD
Ligation tubes (vector:insert 1:5)
[μl] IgG + pSB1C3 SOD + pSB1A3 yCCS A + pSB1A3 yCCS B + pSB1A3
Vector DNA [100 ng] 2.5 2.5 2.5 2.5
Insert DNA [500 ng] 12.5 12.5 12.5 12.5
5X Rapid Ligation buffer 4 4 4 4
T4 DNA ligase 1 1 1 1
  20 20 20 20
  1. Gently mixing
  2. Incubation 22 °C, 10 min
  3. Collection by 5 sec centrifugation
  4. Tubes returned to ice

Quick transformation

  1. 100 μl Top10 cells thawed
  2. 2 μl ligation mixture added to cells. Cells kept on ice ≈5 min
  3. Heat-shock in 42 °C, 30 sec
  4. Cells plated on LB agar with relevant antibiotics
    • 100 Amp
    • 25 Cm
  5. Incubation ON in 37 °C



Mimmi

MITF

the vector pRc/CMV and the insert MITF


















- restriction enzyme

  • the PCR products (from 2010-08-02) are treated with the restriction enzyme AgeI
    • the mutated MITF should show two bands: ~1300bp and ~10bp (which you dont see)
    • the control (non-mutated) MITF should show three bands: ~1060bp, ~200bp and ~10bp (which you don't see)


Mix (µl)
DNA 15
sH2O 2
10X buffer 2
AgeI 1
tot 20


  • Mix and spinn down breafly
  • Incubate 2h (1-16h)


- Gel

  • 0.5% agarose gel
  • (Melted in our bath, redoing the gel in another bath...

Nina

Overnight culture of Tyrosinase

I inoculated Tyrosinase in its original vector from a glycerol stock (that was sent to us from iGEM hq) in 12 ml LB and added 24 ul amphicillin (50 mg/ml). This was incubated in 37 °C overnight in shake. This will be mini prepped tomorrow.





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

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