Team:Stockholm/17 September 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
==Andreas==
==Andreas==
 +
===Plasmid preps===
 +
''From 16/9 ON cultures''
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|-
 +
!Sample
 +
!width="60"|Conc [ng/μl]
 +
!width="60"|A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.N-TAT
 +
|align="center"|64.07
 +
|align="center"|n/a
 +
|-
 +
|pSB1C3.N-Tra10
 +
|align="center"|46.38
 +
|align="center"|n/a
 +
|-
 +
|pSB1C3.N-LMWP
 +
|align="center"|65.77
 +
|align="center"|n/a
 +
|-
 +
|pSB1K3.N-TAT&sdot;SOD&sdot;His 4
 +
|align="center"|87.83
 +
|align="center"|n/a
 +
|-
 +
|pSB1K3.N-TAT&sdot;SOD&sdot;His 5
 +
|align="center"|8.464
 +
|align="center"|n/a
 +
|-
 +
|pSB1K3.N-Tra10&sdot;SOD&sdot;His 5
 +
|align="center"|6.872
 +
|align="center"|n/a
 +
|-
 +
|pEX.SOD
 +
|align="center"|31.57
 +
|align="center"|n/a
 +
|}
 +
 +
 +
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== SOD / yCCS ===
 +
 +
==== over expression ====
 +
 +
*Start culture
 +
**10ml LB<sub>AMP</sub> + 100µl old culture  (8:30)
 +
 +
*Measure OD=0.6  (11:30)
 +
**At OD=0.6 add IPTG 1mM
 +
 +
*Take samples after:
 +
**0h
 +
**2h
 +
***take 500µl, spinn down and remove LB
 +
***resuspend in 50µl SDS buffer
 +
***Heat in 95&deg;C, 5min
 +
***Freeze
 +
***Re-heat in 95&deg;C, 5min
 +
 +
*Run gel
 +
 +
 +
 +
 +
==== PhastGel ====
 +
 +
{|
 +
! well
 +
! sample
 +
| rowspan="9" | [[Image:Place_for_picture.jpg|200px|thumb|left|]]
 +
| rowspan="9" width="50" |
 +
! well
 +
! sample
 +
| rowspan="9" | [[Image:Place_for_picture.jpg|200px|thumb|left|]]
 +
|-
 +
| 1
 +
| ladder
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| SOD.his 0h
 +
| 2
 +
| yCCS 1 0h
 +
|-
 +
| 3
 +
| SOD.his 2h
 +
| 3
 +
| yCCS 1 2h
 +
|-
 +
| 4
 +
| his.SOD 0h
 +
| 4
 +
| yCCS 2 0h
 +
|-
 +
| 5
 +
| his.SOD 2h
 +
| 5
 +
| yCCS 2 2h
 +
|-
 +
| 6
 +
| ladder
 +
| 6
 +
| ladder
 +
|}
 +
 +
 +
=== his.SOD.cTAT ===
 +
 +
==== colony PCR ====
 +
 +
{|
 +
! mix
 +
| (µl)
 +
| x8
 +
| rowspan="8" width="100" |
 +
! Primers
 +
| rowspan="8" width="100" |
 +
! colspan="2" | conditions
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| rowspan="3" |
 +
|-
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| mastermix
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| 24.5
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| rowspan="7" |
 +
| pSB1_VF2
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! time
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! &deg;C
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|-
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| DNA
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| 0.5
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| pSB1_VR
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| 5m
 +
| 95
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|-
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| align="right" | tot
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| 25µl
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| rowspan="5" |
 +
| 30s
 +
| 95
 +
| )
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|-
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| colspan="2" rowspan="4" |
 +
| 30s
 +
| 55
 +
| > 30 cycles
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|-
 +
| 1m20s
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| 72
 +
| )
 +
|-
 +
| 10m
 +
| 72
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| rowspan="2" |
 +
|-
 +
| oo
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| 25
 +
|}
 +
 +
==Johan==
 +
 +
PCR again, to see if the 900 nt-band or the 700 nt-band is correct
 +
 +
0,5 µl pol
 +
 +
0,5 µl dNTP
 +
 +
5 µl 5x buffer
 +
 +
1,5 µl for primer
 +
 +
1,5 µl rev primer
 +
 +
16 µl H2O
 +
 +
{{Stockholm/Footer}}

Latest revision as of 01:37, 28 October 2010

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Contents

Andreas

Plasmid preps

From 16/9 ON cultures

DNA concentration
Sample Conc [ng/μl] A260/A280
pSB1C3.N-TAT 64.07 n/a
pSB1C3.N-Tra10 46.38 n/a
pSB1C3.N-LMWP 65.77 n/a
pSB1K3.N-TAT⋅SOD⋅His 4 87.83 n/a
pSB1K3.N-TAT⋅SOD⋅His 5 8.464 n/a
pSB1K3.N-Tra10⋅SOD⋅His 5 6.872 n/a
pEX.SOD 31.57 n/a




Mimmi

SOD / yCCS

over expression

  • Start culture
    • 10ml LBAMP + 100µl old culture (8:30)
  • Measure OD=0.6 (11:30)
    • At OD=0.6 add IPTG 1mM
  • Take samples after:
    • 0h
    • 2h
      • take 500µl, spinn down and remove LB
      • resuspend in 50µl SDS buffer
      • Heat in 95°C, 5min
      • Freeze
      • Re-heat in 95°C, 5min
  • Run gel



PhastGel

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


his.SOD.cTAT

colony PCR

mix (µl) x8 Primers conditions
mastermix 24.5 pSB1_VF2 time °C
DNA 0.5 pSB1_VR 5m 95
tot 25µl 30s 95 )
30s 55 > 30 cycles
1m20s 72 )
10m 72
oo 25

Johan

PCR again, to see if the 900 nt-band or the 700 nt-band is correct

0,5 µl pol

0,5 µl dNTP

5 µl 5x buffer

1,5 µl for primer

1,5 µl rev primer

16 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/17_September_2010"