Team:Stockholm/14 September 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
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==Andreas==
==Andreas==
===Preparation of Top10 chemically competent cells===
===Preparation of Top10 chemically competent cells===
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*15 min on ice
*15 min on ice
*50 μl IPTG (on pEX.SOD Amp 100 plate only)
*50 μl IPTG (on pEX.SOD Amp 100 plate only)
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== Mimmi ==
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=== MITF-M ===
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==== FB PCR ====
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{|
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! mix
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| (µl)x2
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| rowspan="10" width="100" |
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|
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| rowspan="10" width="100" |
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| colspan="2" |
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| rowspan="4" |
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|-
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| sH<sub>2</sub>O
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| 22.5
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! primers
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! colspan="2" | Conditions
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|-
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| F primer
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| 1
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| MITF_FB_F_18aug
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! time
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! &deg;C
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|-
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| R primer
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| 1
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| MITF_FB_R_18aug
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| 10m
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| 95
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|-
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| DNA
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| 0.5
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| rowspan="6" |
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| 30s
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| 95
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| )
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|-
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| align="right" | tot
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| 25µl
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| 30s
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| 55
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| > 5 cycles
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|-
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| colspan="2" rowspan="4" |
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| 1m40s
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| 72
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| )
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|-
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| 30s
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| 95
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| \
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|-
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| 2m10s
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| 72
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| / 25 cycles
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|-
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| 10m
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| 72
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|}
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==== Digestion ====
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:::- MITF-M and pSB1C3
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{|
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! Mix
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! MITF
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! pSB1C3 cut S
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! pSB1C3 cut E
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| rowspan="7" width="100" |
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! colspan="2" | conditions
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|-
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| sH<sub>2</sub>O
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| 5
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| )
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| )
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! time
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! &deg;C
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|-
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| DNA
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| 20
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| >24
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| >24
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| 30m
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| 37
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|-
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| 10xbuffer
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| 3
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| )
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| )
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| 20m
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| 65
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|-
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| EcoRI
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| 1
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| 1
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|
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| rowspan="3" |
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| rowspan="3" |
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|-
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| SpeI
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| 1
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|
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| 1
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|-
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| align="right" | tot
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| 30µl
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| 25µl
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| 25µl
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|}
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==== Gel ====
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[[Image:2010-09-14_MITF_nu_jaklar.jpg|200px|thumb|left|]]
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{|
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! well
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! sample
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|-
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| 1
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| ladder
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|-
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| 2
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| MITF-M
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|-
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| 3
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| MITF-M
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|-
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| 4
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| MITF-M cut E+S
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|-
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| 5
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| pSB1C3 cut E+S
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|-
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| 6
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| pSB1C3 cut E+S
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|}
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=== SOD.his / his.SOD / yCCS ===
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*preparing for glycerol stock and over-expression...
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{| border="1"
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| SOD.his
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| x2
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| 3ml
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|-
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| his.SOD
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| x2
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| 3ml
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|-
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| yCCS
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| x2
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| 3ml
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|}
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*Grow at 37&deg;C ON (~175rpm)
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{{Stockholm/Footer}}

Latest revision as of 11:03, 26 October 2010

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Contents

Andreas

Preparation of Top10 chemically competent cells

No growth on neither Amp 100, Cm 25 nor Km 50 plates - no contamination of competent cells.

pSB1C3.N-TAT & .N-Tra10

Successful transformation. One colony of each plate picked and dissolved in 10 μl and saved in fridge for later glycerol stock preparation.

Assembly of new parts

Planned and designed cloning strategies for some of the assemblies I will be constructing:

  • pEX.SOD
  • N-TAT⋅SOD⋅His
  • N-Tra10⋅SOD⋅His
  • N-LMWP⋅SOD⋅His (N-LMWP n/a)
  • His⋅SOD⋅C-Tra10 (C-Tra10 n/a)
  • His⋅SOD⋅C-TAT (C-TAT n/a)
  • His⋅SOD⋅C-LMWP (C-LMWP n/a)
  • RBS.yCCS

Digestions

  • [pSB1C3.SOD] = 105.5 ng/μl
  • [pSB1A2.RBS 34] = 60 ng/μl
  • [pMA.His⋅SOD] = 266 ng/μl
  • [pMA.SOD⋅His] = 246 ng/μl
  • [pSB1C3.N-TAT] = 200 ng/μl
  • [pSB1C3.N-Tra10] = 151 ng/μl
  • [pSB1K3.RFP] = 104 ng/μl
  His⋅SOD SOD⋅His RBS 34 pSB1K3 N-TAT N-Tra10 SOD
10X FD buffer 3 3 3 3 3 3 1
dH2O 16.9 17.5 0 5.8 15 11.8 7
DNA 8.1 7.5 25 19.2 10 13.2 1
NgoMIV (NEB) 1 0 0 0 0 0 0
PstI 1 0 1 1 0 0 1
FD SpeI 0 0 1 0 0 0 0
FD EcoRI 0 1 0 1 1 1 0
AgeI 0 1 0 0 1 1 0
FD XbaI 0 0 0 0 0 0 1
  30 μl 30 μl 30 μl 30 μl 30 μl 30 μl 10 μl
  • Incubation: 37 °C
    • 2:00 (conventional enzymes)
    • 0:30 (FastDigest enzymes)
  • Inactivation: 80 °C, 20 min

Ligations

  • [Dig RBS 34 14/9] = 50 ng/μl
  • [Dig pSB1K3 14/9] = 66.6 ng/μl
  • [Dig SOD⋅His 14/9] = 66.6 ng/μl
  • [Dig His⋅SOD 14/9] = 66.6 ng/μl
  • [Dig N-TAT 14/9] = 66.6 ng/μl
  • [Dig N-Tra10 14/9] = 66.6 ng/μl
  • [Dig SOD 14/9] = 15 ng/μl
  Lig pEX.SOD 14/9 Lig pK.N-TAT⋅ SOD⋅His 14/9 Lig pK.N-Tra10⋅ SOD⋅His 14/9 Lig pA.RBS. yCCS 14/9
10X T4 ligase buffer 2 2 2 2
Vector DNA 3 1.5 1.5 2
Insert DNA (1) 10 11 11 6
Insert DNA (2) – 4.5 4.5 –
dH2O 4 0 0 9
T4 DNA ligase 1 1 1 1
  20 μl 20 μl 20 μl 20 μl
  • Incubation: 22 °C, 15 min

Transformation

Standard transformation

  • 1 μl ligation mix
  • 15 min on ice
  • 50 μl IPTG (on pEX.SOD Amp 100 plate only)




Mimmi

MITF-M

FB PCR

mix (µl)x2
sH2O 22.5 primers Conditions
F primer 1 MITF_FB_F_18aug time °C
R primer 1 MITF_FB_R_18aug 10m 95
DNA 0.5 30s 95 )
tot 25µl 30s 55 > 5 cycles
1m40s 72 )
30s 95 \
2m10s 72 / 25 cycles
10m 72


Digestion

- MITF-M and pSB1C3
Mix MITF pSB1C3 cut S pSB1C3 cut E conditions
sH2O 5 ) ) time °C
DNA 20 >24 >24 30m 37
10xbuffer 3 ) ) 20m 65
EcoRI 1 1
SpeI 1 1
tot 30µl 25µl 25µl



Gel

2010-09-14 MITF nu jaklar.jpg
well sample
1 ladder
2 MITF-M
3 MITF-M
4 MITF-M cut E+S
5 pSB1C3 cut E+S
6 pSB1C3 cut E+S




SOD.his / his.SOD / yCCS

  • preparing for glycerol stock and over-expression...
SOD.his x2 3ml
his.SOD x2 3ml
yCCS x2 3ml
  • Grow at 37°C ON (~175rpm)





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

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