Team:Stockholm/17 September 2010

From 2010.igem.org

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== Mimmi ==
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=== SOD / yCCS ===
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==== over expression ====
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*Start culture
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**10ml LB<sub>AMP</sub> + 100µl old culture  (8:30)
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*Measure OD=0.6  (11:30)
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**At OD=0.6 add IPTG 1mM
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*Take samples after:
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**0h
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**2h
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***take 500µl, spinn down and remove LB
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***resuspend in 50µl SDS buffer
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***Heat in 95&deg;C, 5min
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***Freeze
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***Re-heat in 95&deg;C, 5min
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*Run gel
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==== PhastGel ====
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{|
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! well
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! sample
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| rowspan="9" | [[Image:Place_for_picture.jpg|200px|thumb|left|]]
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| rowspan="9" width="50" |
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! well
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! sample
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| rowspan="9" | [[Image:Place_for_picture.jpg|200px|thumb|left|]]
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|-
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| 1
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| ladder
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| 1
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| ladder
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|-
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| 2
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| SOD.his 0h
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| 2
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| yCCS 1 0h
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|-
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| 3
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| SOD.his 2h
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| 3
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| yCCS 1 2h
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|-
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| 4
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| his.SOD 0h
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| 4
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| yCCS 2 0h
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|-
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| 5
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| his.SOD 2h
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| 5
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| yCCS 2 2h
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|-
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| 6
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| ladder
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| 6
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| ladder
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|}
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=== his.SOD.cTAT ===
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==== colony PCR ====
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{|
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! mix
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| (µl)
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| x8
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| rowspan="8" width="100" |
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! Primers
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| rowspan="8" width="100" |
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! colspan="2" | conditions
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| rowspan="3" |
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|-
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| mastermix
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| 24.5
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| rowspan="7" |
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| 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
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| 95
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|-
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| align="right" | tot
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| 25µl
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| rowspan="5" |
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| 30s
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| 95
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| )
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|-
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| colspan="2" rowspan="4" |
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| 30s
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| 55
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| > 30 cycles
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|-
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| 1m20s
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| 72
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| )
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|-
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| 10m
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| 72
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| rowspan="2" |
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|-
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| oo
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| 25
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|}
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==Johan==
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PCR again, to see if the 900 nt-band or the 700 nt-band is correct
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0,5 µl pol
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0,5 µl dNTP
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5 µl 5x buffer
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1,5 µl for primer
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1,5 µl rev primer
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16 µl H2O
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{{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"