Team:Stockholm/15 September 2010

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==Andreas==
==Andreas==
 +
===Assembly of new parts===
 +
====Transformation results====
 +
Good (white) colony yield on all plates. Picked 4 from each for colony PCR.
 +
 +
====Colony PCR====
 +
*pSB1K3.N-TAT⋅SOD⋅His: TAT.SH 1-4
 +
*pSB1K3.N-Tra10⋅SOD⋅His: Tra10 SH 1-4
 +
*pSB1A2.RBS.yCCS: RBS.y 1-4
 +
*pEX.SOD 1-4
 +
''Controls''
 +
*pEX.RFP
 +
*pSB1A2.RBS
 +
*pSB1C3.SOD
 +
*pEX.SOD⋅His
 +
*pSB1C3.SOD⋅His
 +
 +
Standard colony PCR settings
 +
*Elongation: 1:20
 +
 +
====Gel verification====
 +
=====Gel 1=====
 +
[[image:ColPCR_TAT-Tra10*SH_15sep.png|200px|thumb|right|'''Colony PCR gel verification of N-TAT&sdot;SOD&sdot;His and N-Tra10&sdot;SOD&sdot;His assemblies in pSB1K3.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = GeneRuler 1 kb DNA ladder.]]
 +
1 % agarose, 90 V
 +
 +
'''Expected bands'''
 +
*pSB1K3.N-TAT&sdot;SOD&sdot;His: 854 bp
 +
*pSB1K3.N-Tra10&sdot;SOD&sdot;His: 884 bp
 +
*pSB1C3.SOD&sdot;His: 815 bp
 +
 +
'''Results'''<br />
 +
Very unclear gel. Ladders are impossible to read. Two N-Tra10 clones (Tra10 SH1 and 2) seem to have migrated slightly slower than the SOD&sdot;His control, which may be a sign of successful insertion of Tra10. PCR samples will be analyzed again tomorrow.
 +
 +
=====Gel 2=====
 +
[[image:ColPCR_RBS.yCCS_15sep.png|200px|thumb|right|'''Colony PCR gel verification of RBS(BBa_B0034).yCCS assemblies in pSB1A2.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = GeneRuler 1 kb DNA ladder]]
 +
1 % agarose, 90 V
 +
 +
'''Expected bands'''
 +
*pSB1A2.RBS.yCCS: 1018 bp
 +
*pSB1A2.RBS: 250 bp
 +
 +
'''Results'''<br />
 +
All bands at 250 bp, clearly showing that insertion of yCCS was unsuccessful. Incomplete digestion of pSB1A2.RBS with SpeI and PstI might be the cause of problem.
 +
 +
=====Gel 3=====
 +
[[image:ColPCR_pEX.SOD_15sep.png|200px|thumb|right|'''Colony PCR gel verification of pEX.SOD.'''<br />4 &mu;l &lambda;; 5 &mu;l sample.<br />&lambda; = GeneRuler 1 kb DNA ladder]]
 +
1 % agarose, 90 V
 +
 +
'''Expected bands'''
 +
*pEX.SOD: 678 bp
 +
*pEX.RFP: 1385 bp
 +
*pEX.SOD&sdot;His: 702 bp
 +
 +
'''Results'''<br />
 +
No pEX.SOD bands whatsoever, probably due to lack of template in PCR tubes. Will re-run colony PCR tomorrow.
 +
 +
===Plasmid prep===
 +
''From Johan's ON culture''
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|-
 +
!Sample
 +
!Conc [ng/&mu;l]
 +
!A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pSB1C3.C-TAT
 +
|align="center"|123.7
 +
|align="center"|1.82
 +
|}
 +
 +
===Assembly of His&sdot;SOD&sdot;C-TAT===
 +
====Digestion====
 +
 +
[pSB1C3.C-TAT] = 123.7 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
|10X FastDigest buffer
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|align="center"|3
 +
|-
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|dH<sub>2</sub>O
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|align="center"|8.8
 +
|-
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|DNA (2 &mu;g)
 +
|align="center"|16.2
 +
|-
 +
|NgoMIV
 +
|align="center"|1
 +
|-
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|FD PstI
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|align="center"|1
 +
|-
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|&nbsp;
 +
!30 &mu;l
 +
|}
 +
*Incubation: 37 &deg;C, 2:00 (NgoMIV); 0:30 (FD PstI)
 +
*Inactivation: 80 &deg;C, 20 min
 +
 +
====Ligation====
 +
 +
*Vector: [Dig pSB1K3.RFP E+P 14/9] = 66.6 ng/&mu;l
 +
*Insert 1: [Dig pMA.His&sdot;SOD E+A 14/9] = 66.6 ng/&mu;l
 +
*Insert 2: [Dig pSB1C3.C-TAT N+P 15/9] = 66.6 ng/&mu;l
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
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|10X T4 Ligase buffer
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|align="center"|2
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|-
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|dH<sub>2</sub>O
 +
|align="center"|0
 +
|-
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|Vector DNA
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|align="center"|1.5
 +
|-
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|Insert 1 DNA
 +
|align="center"|4.5
 +
|-
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|Insert 2 DNA
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|align="center"|11
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|-
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|T4 DNA ligase
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|align="center"|1
 +
|-
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|&nbsp;
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!20 &mu;l
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|}
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*Incubation: 22 &deg;C, 15 min
 +
 +
====Transformation====
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Standard transformation
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*1 &mu;l ligation mix
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*Km 50 plate
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 +
===ON cultures===
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*3 ml LB + Cm 25; 30 &deg;C
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**pSB1C3.C-TAT
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*5 ml LB + Cm 25; 37 &deg;C, 225 rpm
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**pSB1C3.N-TAT
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**pSB1C3.N-Tra10
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**pSB1C3.SOD
 +
 +
 +
 +
 +
 +
 +
 +
 +
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== Mimmi ==
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 +
=== SOD.his / his.SOD / yCCS ===
 +
 +
==== glycerol stock / over expression ====
 +
 +
{|
 +
|
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| glycerol stock
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| over expression
 +
|
 +
|-
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| SOD.his x2
 +
|  2ml +  10µl
 +
| 10ml + 100µl
 +
| *Start growing 9:30
 +
|-
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| his.SOD x2
 +
|  2ml +  10µl
 +
| 10ml + 100µl
 +
| rowspan="2" |
 +
|-
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| yCCS x2
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|  2ml +  10µl
 +
| 10ml + 100µl
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|}
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 +
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*At OD=0.6 add IPTG /make glycerol stock  (14:30)
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**Sample 0h
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***1ml culture
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***Spinn down 13000rpm, 5min, remove LB
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***Resuspend in 100µl sample buffer
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***Heat in 95&deg;C, 5min
 +
***Freeze
 +
 +
*Sample 1h
 +
 +
*Sample 2h
 +
 +
*Sample 3h
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 +
{{Stockholm/Footer}}

Latest revision as of 11:03, 26 October 2010

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Contents

Andreas

Assembly of new parts

Transformation results

Good (white) colony yield on all plates. Picked 4 from each for colony PCR.

Colony PCR

  • pSB1K3.N-TAT⋅SOD⋅His: TAT.SH 1-4
  • pSB1K3.N-Tra10⋅SOD⋅His: Tra10 SH 1-4
  • pSB1A2.RBS.yCCS: RBS.y 1-4
  • pEX.SOD 1-4

Controls

  • pEX.RFP
  • pSB1A2.RBS
  • pSB1C3.SOD
  • pEX.SOD⋅His
  • pSB1C3.SOD⋅His

Standard colony PCR settings

  • Elongation: 1:20

Gel verification

Gel 1
Colony PCR gel verification of N-TAT⋅SOD⋅His and N-Tra10⋅SOD⋅His assemblies in pSB1K3.
4 μl λ; 5 μl sample.
λ = GeneRuler 1 kb DNA ladder.

1 % agarose, 90 V

Expected bands

  • pSB1K3.N-TAT⋅SOD⋅His: 854 bp
  • pSB1K3.N-Tra10⋅SOD⋅His: 884 bp
  • pSB1C3.SOD⋅His: 815 bp

Results
Very unclear gel. Ladders are impossible to read. Two N-Tra10 clones (Tra10 SH1 and 2) seem to have migrated slightly slower than the SOD⋅His control, which may be a sign of successful insertion of Tra10. PCR samples will be analyzed again tomorrow.

Gel 2
Colony PCR gel verification of RBS(BBa_B0034).yCCS assemblies in pSB1A2.
4 μl λ; 5 μl sample.
λ = GeneRuler 1 kb DNA ladder

1 % agarose, 90 V

Expected bands

  • pSB1A2.RBS.yCCS: 1018 bp
  • pSB1A2.RBS: 250 bp

Results
All bands at 250 bp, clearly showing that insertion of yCCS was unsuccessful. Incomplete digestion of pSB1A2.RBS with SpeI and PstI might be the cause of problem.

Gel 3
Colony PCR gel verification of pEX.SOD.
4 μl λ; 5 μl sample.
λ = GeneRuler 1 kb DNA ladder

1 % agarose, 90 V

Expected bands

  • pEX.SOD: 678 bp
  • pEX.RFP: 1385 bp
  • pEX.SOD⋅His: 702 bp

Results
No pEX.SOD bands whatsoever, probably due to lack of template in PCR tubes. Will re-run colony PCR tomorrow.

Plasmid prep

From Johan's ON culture

DNA concentration
Sample Conc [ng/μl] A260/A280
pSB1C3.C-TAT 123.7 1.82

Assembly of His⋅SOD⋅C-TAT

Digestion

[pSB1C3.C-TAT] = 123.7 ng/μl

10X FastDigest buffer 3
dH2O 8.8
DNA (2 μg) 16.2
NgoMIV 1
FD PstI 1
  30 μl
  • Incubation: 37 °C, 2:00 (NgoMIV); 0:30 (FD PstI)
  • Inactivation: 80 °C, 20 min

Ligation

  • Vector: [Dig pSB1K3.RFP E+P 14/9] = 66.6 ng/μl
  • Insert 1: [Dig pMA.His⋅SOD E+A 14/9] = 66.6 ng/μl
  • Insert 2: [Dig pSB1C3.C-TAT N+P 15/9] = 66.6 ng/μl
10X T4 Ligase buffer 2
dH2O 0
Vector DNA 1.5
Insert 1 DNA 4.5
Insert 2 DNA 11
T4 DNA ligase 1
  20 μl
  • Incubation: 22 °C, 15 min

Transformation

Standard transformation

  • 1 μl ligation mix
  • Km 50 plate

ON cultures

  • 3 ml LB + Cm 25; 30 °C
    • pSB1C3.C-TAT
  • 5 ml LB + Cm 25; 37 °C, 225 rpm
    • pSB1C3.N-TAT
    • pSB1C3.N-Tra10
    • pSB1C3.SOD






Mimmi

SOD.his / his.SOD / yCCS

glycerol stock / over expression

glycerol stock over expression
SOD.his x2 2ml + 10µl 10ml + 100µl *Start growing 9:30
his.SOD x2 2ml + 10µl 10ml + 100µl
yCCS x2 2ml + 10µl 10ml + 100µl


  • At OD=0.6 add IPTG /make glycerol stock (14:30)
    • Sample 0h
      • 1ml culture
      • Spinn down 13000rpm, 5min, remove LB
      • Resuspend in 100µl sample buffer
      • Heat in 95°C, 5min
      • Freeze
  • Sample 1h
  • Sample 2h
  • Sample 3h





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

Retrieved from "http://2010.igem.org/Team:Stockholm/15_September_2010"