Team:Stockholm/27 July 2010

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[[Image:Net image a jM9yggVoo68J high res.jpg|400px|thumb|left|Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6]]
+
 
 +
== Mimmi ==
 +
 
 +
 
 +
=== over-expression in pEX ===
 +
::::- '''remaking the comassie-gel'''
 +
 
 +
 
 +
 
 +
{| border="1" cellspacing="0"
 +
|-
 +
! Mix
 +
| v
 +
|-
 +
| LB
 +
| 4 ml
 +
|-
 +
| glycose
 +
| 40 µl (1%)
 +
|-
 +
| Amp
 +
| 8 µl (0.5%)
 +
|-
 +
| old culture
 +
| 40 µl
 +
|}
 +
 
 +
 
 +
*Grow in 37C, ~200 rpm ~2h until OD = 0.6
 +
 
 +
*Add IPTG (1 µl/ml,1M)in one of the two cultures, the other one is used as a control
 +
 
 +
*Take sample at 0h, 1h, 2h, 3h
 +
**pipette 500µl into a 1.5ml tube
 +
**spinn down the sells and remove LB
 +
**resuspend in 50µl loading dye
 +
**freeze
 +
 
 +
 
 +
 
 +
=== Site-Directed Mutagenesis ===
 +
 
 +
==== Primers ====
 +
:(MW/10)/(OD*33) = dilution factor
 +
::1000/df = volyme to get 100µM primer
 +
:::100µM = (MW/10) ng/µl
 +
::::(MW/10)/125 = df to get 125 ng/µl
 +
 
 +
::{| border="1" cellspacing="0"
 +
|-
 +
! primer
 +
! V H2O (µl)
 +
! V H2O to get 125 ng/µl (µl)
 +
|-
 +
| MITF_site1_F
 +
| 463.88
 +
| 3µl + 15.4µl
 +
|-
 +
| MITF_site1_R
 +
| 64.77
 +
| 3µl + 15.3µl
 +
|-
 +
| MITF_site2_F
 +
| 144.85
 +
| 3µl + 21µl
 +
|-
 +
| MITF_site2_R
 +
| 141.77
 +
| 3µl + 21.6µl
 +
|-
 +
|
 +
! concentration
 +
|
 +
|-
 +
| yCCS_F
 +
| 1085.7 ng/µl
 +
| 3µl + 23µl
 +
|-
 +
| yCCS_R
 +
| 996.5 ng/µl
 +
| 3µl + 20.9µl
 +
|}
 +
 
 +
==== reaction ====
 +
 
 +
{| border="1" cellspacing="0"
 +
|-
 +
! Mix
 +
| width="50" | (µl)
 +
| width="50" |X2
 +
|-
 +
| H2O
 +
| 40
 +
| 80
 +
|-
 +
| dNTPs
 +
| 1
 +
| 2
 +
|-
 +
| F primer
 +
| 1
 +
| 2
 +
|-
 +
| R primer
 +
| 1
 +
| 2
 +
|-
 +
| DNA
 +
| 1
 +
| 2*1
 +
|-
 +
| Pfu X10 buffer
 +
| 5
 +
| 10
 +
|-
 +
| Pfu turbo pol
 +
| 1
 +
| 2
 +
|}
 +
:::::::{| border="0" width="50%" |
 +
! align="left" | MITF
 +
! align="left" | yCCS
 +
! conditions
 +
|-
 +
|MITF_Site1_F
 +
|yCCS_F
 +
!align="left" width="1%"|time
 +
!align="left" width="1%"|°C
 +
|-
 +
|MITF_Site2_R
 +
|yCCS_R
 +
|30s
 +
|95
 +
|-
 +
|pRc/CMV
 +
|pSB1C3
 +
|30s
 +
|95
 +
|-
 +
|~6.7kb
 +
|~3,5kb
 +
|30s
 +
|55
 +
|-
 +
|
 +
|
 +
|7m
 +
|68
 +
|-
 +
|
 +
|
 +
|oo
 +
|4
 +
|}
 +
 
 +
 
 +
::*Make sure sample is ≤ 37°C
 +
::*Add 1µl Dpn1 (to 50µl product) and incubate in 37°C ON
 +
 
 +
 
 +
 
 +
===Transformation===
 +
 
 +
::<u>mix</u>
 +
::Top 10 competent cells 100µl
 +
::pRc/CMV.MITF_M 1µl
 +
 
 +
 
 +
*Hold on ice 30min
 +
*Heat shock 42&degC, 55sec
 +
*Cool down 1min (on ice)
 +
*Add 900µl LB
 +
*Incubate 37&deg;C, 250rpm, 1h (forgot rpm)
 +
*Spinn down cells, 13000rpm, 15sec
 +
*Remove 900µl LB
 +
*Plate the 100µl on a Amp-plate
 +
*Grow ON at 37&deg;C
 +
 
 +
==Hassan==
 +
 
 +
[[Image:Net image a jM9yggVoo68J high res.jpg|500px|thumb|left|Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6]]
[[Image:Net image a V5uyW3lEbFAM.jpg|400px|thumb|left|Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6]]
[[Image:Net image a V5uyW3lEbFAM.jpg|400px|thumb|left|Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6]]
 +
----
 +
 +
[http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000364016%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&interactive=yes&network_flavor=actions&targetmode=proteins]
 +
 +
[http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins]
 +
 +
 +
----
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 +
----
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----
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----
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----
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----
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----
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----
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----
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----
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----
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 +
----
 +
 +
==Nina==
 +
 +
==Colony PCR on IgG protease in shipping vector==
 +
 +
I made a colony PCR on the IgG protease that I have inserted into the iGEM shipping vector to verify that the gene is inserted in a correct position. Therefore I used one of the gene's primers and one of the vector's verification primers. The colony numbers are: 1, 2, 3, 4 and 5.
 +
 +
PCR reaction mix:
 +
 +
* 1 µl Morten's polymerase PjuX7
 +
 +
* 1 µl 10 mM dNTPs
 +
 +
* 3 µl 5 µM forward primer (VF2)
 +
 +
* 3 µl 5 µM revers primer (gene's primer)
 +
 +
* 10 µl buffer 5X
 +
 +
* 1 µl MgCl2 50mM
 +
 +
* 30 µl H2O
 +
 +
* DNA template was one colony 
 +
 +
PCR program:
 +
 +
98°C - 2 min
 +
 +
31 cycles of:
 +
 +
* 98°C - 10 sec
 +
 +
* 55°C - 15 sec
 +
 +
* 72°C - 1.5 min
 +
 +
72°C - 5 min
 +
 +
4°C - ∞
 +
 +
[[Image:Igg-shipping.jpg|250px]]
 +
 +
DNA Ladder: FastRuler™ Middle Range, ready-to-use, 100-5000 bp Fermentas
 +
 +
Colony number 2 looks good on the gel. This one will be inoculated in LB in order to become minipreped to be shipped to iGEM hq.
 +
 +
----
 +
 +
==Mini prep on IgG protease and CPP==
 +
 +
I prepare a miniprep on the inoculated IgG protease with colony number 2. In addition I miniprep three inoculated samples with vector carrying CPP from colony number 22, 23 and 33.
 +
 +
The method is carried out according to the procedure in protocols.
 +
 +
Measuring concentration with spectrophotometer:
 +
 +
[[Image:Spek.jpg]]
 +
 +
----
 +
 +
==Sequencing CPP TAT N version==
 +
 +
I send three samples of CPP TAT N version for sequencing. Colony numbers are: 22, 23 and 33.
 +
 +
* 15 ul vector
 +
 +
* 1.5 ul 10uM VR2 primer
 +
 +
22: ASB0045 105
 +
 +
23: ASB0045 104
 +
 +
33: ASB0045 103
-
[http://http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins]
+
{{Stockholm/Footer}}

Latest revision as of 10:56, 26 October 2010

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Contents

Mimmi

over-expression in pEX

- remaking the comassie-gel


Mix v
LB 4 ml
glycose 40 µl (1%)
Amp 8 µl (0.5%)
old culture 40 µl


  • Grow in 37C, ~200 rpm ~2h until OD = 0.6
  • Add IPTG (1 µl/ml,1M)in one of the two cultures, the other one is used as a control
  • Take sample at 0h, 1h, 2h, 3h
    • pipette 500µl into a 1.5ml tube
    • spinn down the sells and remove LB
    • resuspend in 50µl loading dye
    • freeze


Site-Directed Mutagenesis

Primers

(MW/10)/(OD*33) = dilution factor
1000/df = volyme to get 100µM primer
100µM = (MW/10) ng/µl
(MW/10)/125 = df to get 125 ng/µl
primer V H2O (µl) V H2O to get 125 ng/µl (µl)
MITF_site1_F 463.88 3µl + 15.4µl
MITF_site1_R 64.77 3µl + 15.3µl
MITF_site2_F 144.85 3µl + 21µl
MITF_site2_R 141.77 3µl + 21.6µl
concentration
yCCS_F 1085.7 ng/µl 3µl + 23µl
yCCS_R 996.5 ng/µl 3µl + 20.9µl

reaction

Mix (µl) X2
H2O 40 80
dNTPs 1 2
F primer 1 2
R primer 1 2
DNA 1 2*1
Pfu X10 buffer 5 10
Pfu turbo pol 1 2
MITF yCCS conditions
MITF_Site1_F yCCS_F time °C
MITF_Site2_R yCCS_R 30s 95
pRc/CMV pSB1C3 30s 95
~6.7kb ~3,5kb 30s 55
7m 68
oo 4


  • Make sure sample is ≤ 37°C
  • Add 1µl Dpn1 (to 50µl product) and incubate in 37°C ON


Transformation

mix
Top 10 competent cells 100µl
pRc/CMV.MITF_M 1µl


  • Hold on ice 30min
  • Heat shock 42&degC, 55sec
  • Cool down 1min (on ice)
  • Add 900µl LB
  • Incubate 37°C, 250rpm, 1h (forgot rpm)
  • Spinn down cells, 13000rpm, 15sec
  • Remove 900µl LB
  • Plate the 100µl on a Amp-plate
  • Grow ON at 37°C

Hassan

Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6
Jensen et al. Nucleic Acids Res. 2009, 37(Database issue):D412-6

[http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000364016%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&interactive=yes&network_flavor=actions&targetmode=proteins]

[http://string.embl.de/version_8_3/newstring_cgi/show_network_section.pl?identifiers=9606.ENSP00000331746%250D9606.ENSP00000295600%250D9606.ENSP00000226877%250D9606.ENSP00000371175%250D9606.ENSP00000269280%250D9606.ENSP00000363700%250D9606.ENSP00000360157%250D9606.ENSP00000373571%250D9606.ENSP00000291700&channel1=off&channel2=off&channel3=off&channel4=on&channel5=on&channel6=on&channel7=on&additional_network_nodes=5&interactive=yes&network_flavor=actions&targetmode=proteins]








































Nina

Colony PCR on IgG protease in shipping vector

I made a colony PCR on the IgG protease that I have inserted into the iGEM shipping vector to verify that the gene is inserted in a correct position. Therefore I used one of the gene's primers and one of the vector's verification primers. The colony numbers are: 1, 2, 3, 4 and 5.

PCR reaction mix:

  • 1 µl Morten's polymerase PjuX7
  • 1 µl 10 mM dNTPs
  • 3 µl 5 µM forward primer (VF2)
  • 3 µl 5 µM revers primer (gene's primer)
  • 10 µl buffer 5X
  • 1 µl MgCl2 50mM
  • 30 µl H2O
  • DNA template was one colony

PCR program:

98°C - 2 min

31 cycles of:

  • 98°C - 10 sec
  • 55°C - 15 sec
  • 72°C - 1.5 min

72°C - 5 min

4°C - ∞

Igg-shipping.jpg

DNA Ladder: FastRuler™ Middle Range, ready-to-use, 100-5000 bp Fermentas

Colony number 2 looks good on the gel. This one will be inoculated in LB in order to become minipreped to be shipped to iGEM hq.

Mini prep on IgG protease and CPP

I prepare a miniprep on the inoculated IgG protease with colony number 2. In addition I miniprep three inoculated samples with vector carrying CPP from colony number 22, 23 and 33.

The method is carried out according to the procedure in protocols.

Measuring concentration with spectrophotometer:

Spek.jpg

Sequencing CPP TAT N version

I send three samples of CPP TAT N version for sequencing. Colony numbers are: 22, 23 and 33.

  • 15 ul vector
  • 1.5 ul 10uM VR2 primer

22: ASB0045 105

23: ASB0045 104

33: ASB0045 103





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

Retrieved from "http://2010.igem.org/Team:Stockholm/27_July_2010"