Team:Stockholm/17 September 2010

From 2010.igem.org

(Difference between revisions)
(Plasmid preps=)
Line 38: Line 38:
|align="center"|31.57
|align="center"|31.57
|align="center"|n/a
|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
 +
| rowspan="3" |
 +
|-
 +
| mastermix
 +
| 24.5
 +
| rowspan="7" |
 +
| pSB1_VF2
 +
! time
 +
! &deg;C
 +
|-
 +
| DNA
 +
| 0.5
 +
| pSB1_VR
 +
| 5m
 +
| 95
 +
|-
 +
| align="right" | tot
 +
| 25µl
 +
| rowspan="5" |
 +
| 30s
 +
| 95
 +
| )
 +
|-
 +
| colspan="2" rowspan="4" |
 +
| 30s
 +
| 55
 +
| > 30 cycles
 +
|-
 +
| 1m20s
 +
| 72
 +
| )
 +
|-
 +
| 10m
 +
| 72
 +
| rowspan="2" |
 +
|-
 +
| oo
 +
| 25
|}
|}

Revision as of 12:45, 4 October 2010


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