Team:Stockholm/19 October 2010

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{{Stockholm/Top2}}
{{Stockholm/Top2}}
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==Nina==
 +
 +
===Agarose gel on Colony PCR===
 +
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I ran agarose gels on all the colony PCR screens from yesterday to check if I had inserts.
 +
 +
[[Image:Aq1.jpg|200px]]
 +
 +
[[Image:Aq2.jpg|200px]]
 +
 +
[[Image:Aq3.jpg|200px]]
 +
 +
[[Image:Aq4.jpg|200px]]
 +
 +
Unfortunately non of these gels show that there has been an proper insert of Fusion and Protein A all with the three CPPs in the N-terminal in the pEX vector.
 +
 +
I made a bad decision to conduct the ligation of these parts with the pEX vector and transform the ligations directly into overexpression cells such as BL21. I should have transformed the ligations into cloning cells such as Top 10 and from there as usual perform a mini prep and transform the vectors with insert into BL21 cells. However I hoped some of the ligations that would have ended up as vectors with insert would succesfully get transformed into BL21, but it seems from the gels that this has not happened. We are short of time in the competition and this was a test to see if I could skip the two days I had to spend with transformation into Top 10, but it unfortunatelly did not work out this time. 
 +
 +
 +
 +
 +
 +
 +
 +
==Andreas==
==Andreas==
Line 30: Line 56:
Incorrect size. Send to the Uppsala team for analysis.
Incorrect size. Send to the Uppsala team for analysis.
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-
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-
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-
==Nina==
 
-
===Agarose gel on Colony PCR===
 
-
I ran agarose gels on all the colony PCR screens from yesterday to check if I had inserts.
 
-
[[Image:Aq1.jpg|200px]]
 
-
[[Image:Aq2.jpg|200px]]
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
== Mimmi ==
 +
 
 +
=== SOD activity ===
 +
-continuing
 +
 
 +
 
 +
 
 +
*Sonicate 3x30s at ~12amp
 +
 
 +
*Make solutions
 +
**WST working solution 20ml
 +
**Enzyme working solution 2.5ml
 +
 
 +
 
 +
 
 +
{|
 +
! mix
 +
| samples
 +
| blank 1
 +
| blank 2
 +
| blank 3
 +
|-
 +
| sample solution
 +
| 20
 +
|
 +
| 20
 +
|
 +
|-
 +
| ddH<sub>2</sub>O
 +
|
 +
| 20
 +
|
 +
| 20
 +
|-
 +
| WST solution
 +
| 200
 +
| 200
 +
| 200
 +
| 200
 +
|-
 +
| Enzyme solution
 +
| 20
 +
| 20
 +
|
 +
|
 +
|-
 +
| dilution buffer
 +
|
 +
|
 +
| 20
 +
| 20
 +
|-
 +
| align="right" | tot
 +
| 240µl
 +
| 240µl
 +
| 240µl
 +
| 240µl
 +
|}
 +
 
 +
*Incubate in 37&deg;C for 20 min
 +
 
 +
*Measure A<sub>440</sub> with nanodrop
 +
 
 +
 
 +
*SOD activity (inhibition %) = ((A<sub>blank1</sub> - A<sub>blank3</sub>) - (A<sub>sample</sub> - A<sub>blank2</sub>))/(A<sub>blank1</sub> - A<sub>blank3</sub>) x 100
 +
 
 +
[[Image:SOD_activity1.jpg| 900px]]
 +
 
 +
 
 +
==== cultures ====
 +
Top10
 +
*pEX.SOD.his.RBS.yCCS 1
 +
*pEX.SOD.his.RBS.yCCS 1
 +
BL21
 +
*pEX.SOD.RBS.yCCS
 +
*pEX.his.SOD.RBS.yCCS
 +
 
 +
 
 +
=== ON cultures ===
 +
{|
 +
| pSB1C3.nLMWP
 +
| )
 +
|-
 +
| pSB1C3.nTAT
 +
|  \ plasmid prep.
 +
|-
 +
| pSB1C3.nTra10
 +
|  /
 +
|-
 +
| pEX.SOD.his.RBS.yCCS
 +
| )
 +
|-
 +
| pEX.SOD
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| \ SOD activity test
 +
|-
 +
| pEX.yCCS
 +
| /
 +
|-
 +
| pEX.bFGF
 +
|  )
 +
|-
 +
| pEX.IgGp
 +
| /
 +
|-
 +
| pEX.Prot.A
 +
| > glycerol stock
 +
|-
 +
| pEX.SOD
 +
| \
 +
|-
 +
| pEX.yCCS
 +
|  )
 +
|}
 +
 
 +
=Johan=
 +
 
 +
==Miniprep==
 +
3 ml of sampled "5-6", "5-8", "AS10" and "EA3". Verification of the last constructs, his-bFGF, bFGF-his and tat-bFGF-his.
 +
 
 +
==Cut miniprep==
 +
 
 +
5 µl DNA
 +
 
 +
(1 µl BamHI enzyme)
 +
 
 +
2 µl 10x buffer
 +
 
 +
12 µl H2O
 +
 
 +
==PCR screen==
 +
Of the other constructs in BL21 cells, tra10-bFGF-his, lmwp-bfgf-his, his-bFGF-tra10, his-bFGF-tat, his-bFGF-lmwp. 2 colonies for each plate.
 +
 
 +
0,5 µl Taq pol
 +
 
 +
0,5 µl dNTP
 +
 
 +
5 µl 5x buffer
 +
 
 +
1 µl pex for primer
 +
 
 +
1 µl pex rev primer
 +
 
 +
17 µl H2O
 +
 
 +
A mastermix 10x was made
 +
 
 +
==Gel of minipreps and PCR screen==
 +
 
 +
Top lanes: Cut & uncut miniprep
 +
 
 +
Bottom lanes: PCR screens
 +
 
 +
[[Image:SU 19oktgel.png]]
 +
 
 +
Results:
 +
 
 +
Top lanes, I realized I put BamHI even in the samples that was supposed to be uncut!
 +
 
 +
Bottom lanes: Correct size in the first lane but otherwise empty, why?
 +
 
 +
{{Stockholm/Footer}}

Latest revision as of 19:03, 27 October 2010

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Contents

Nina

Agarose gel on Colony PCR

I ran agarose gels on all the colony PCR screens from yesterday to check if I had inserts.

Aq1.jpg

Aq2.jpg

Aq3.jpg

Aq4.jpg

Unfortunately non of these gels show that there has been an proper insert of Fusion and Protein A all with the three CPPs in the N-terminal in the pEX vector.

I made a bad decision to conduct the ligation of these parts with the pEX vector and transform the ligations directly into overexpression cells such as BL21. I should have transformed the ligations into cloning cells such as Top 10 and from there as usual perform a mini prep and transform the vectors with insert into BL21 cells. However I hoped some of the ligations that would have ended up as vectors with insert would succesfully get transformed into BL21, but it seems from the gels that this has not happened. We are short of time in the competition and this was a test to see if I could skip the two days I had to spend with transformation into Top 10, but it unfortunatelly did not work out this time.





Andreas

Colony PCRs

  • pEX.ProtA⋅His: PA 1 & 2
  • pEX.IgGp: Ig 1 & 2

Standard colony PCR settings

  • Elongation time: 1:20

Gel verification

Colony PCR of pEX.ProtA*His and pEX.IgGp constructs.
3 μl λ;5 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

Expected bands

  • PA: 417 bp
  • Ig: 1149 bp

Results
All four constructs confirmed.

PCR for Uppsala team

Gel verification

PCR run for the Uppsala team.
3 μl λ; 5 μl sample
λ = O'GeneRuler 1 kb DNA ladder.

Expected bands

  • All constructs: ≈ 6570 bp

Results
Incorrect size. Send to the Uppsala team for analysis.















Mimmi

SOD activity

-continuing


  • Sonicate 3x30s at ~12amp
  • Make solutions
    • WST working solution 20ml
    • Enzyme working solution 2.5ml


mix samples blank 1 blank 2 blank 3
sample solution 20 20
ddH2O 20 20
WST solution 200 200 200 200
Enzyme solution 20 20
dilution buffer 20 20
tot 240µl 240µl 240µl 240µl
  • Incubate in 37°C for 20 min
  • Measure A440 with nanodrop


  • SOD activity (inhibition %) = ((Ablank1 - Ablank3) - (Asample - Ablank2))/(Ablank1 - Ablank3) x 100

SOD activity1.jpg


cultures

Top10

  • pEX.SOD.his.RBS.yCCS 1
  • pEX.SOD.his.RBS.yCCS 1

BL21

  • pEX.SOD.RBS.yCCS
  • pEX.his.SOD.RBS.yCCS


ON cultures

pSB1C3.nLMWP )
pSB1C3.nTAT \ plasmid prep.
pSB1C3.nTra10 /
pEX.SOD.his.RBS.yCCS )
pEX.SOD \ SOD activity test
pEX.yCCS /
pEX.bFGF )
pEX.IgGp /
pEX.Prot.A > glycerol stock
pEX.SOD \
pEX.yCCS )

Johan

Miniprep

3 ml of sampled "5-6", "5-8", "AS10" and "EA3". Verification of the last constructs, his-bFGF, bFGF-his and tat-bFGF-his.

Cut miniprep

5 µl DNA

(1 µl BamHI enzyme)

2 µl 10x buffer

12 µl H2O

PCR screen

Of the other constructs in BL21 cells, tra10-bFGF-his, lmwp-bfgf-his, his-bFGF-tra10, his-bFGF-tat, his-bFGF-lmwp. 2 colonies for each plate.

0,5 µl Taq pol

0,5 µl dNTP

5 µl 5x buffer

1 µl pex for primer

1 µl pex rev primer

17 µl H2O

A mastermix 10x was made

Gel of minipreps and PCR screen

Top lanes: Cut & uncut miniprep

Bottom lanes: PCR screens

SU 19oktgel.png

Results:

Top lanes, I realized I put BamHI even in the samples that was supposed to be uncut!

Bottom lanes: Correct size in the first lane but otherwise empty, why?





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

Retrieved from "http://2010.igem.org/Team:Stockholm/19_October_2010"