Team:Stockholm/6 October 2010

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==Andreas==
==Andreas==
 +
 +
===Sequencing results===
 +
Received sequencing results for the following samples ([[media:Sequencings_fasta_6oct.txt|fasta]]):
 +
*pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 1 (VF2 & VR)
 +
*pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
 +
*pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
 +
*pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR)
 +
*pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
 +
*pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR)
 +
*pEX.nTra10⋅SOD⋅His (pEXf) ([[media:PEX.nT10SH_VF_premix_fasta_6oct.txt|fasta]])
 +
*pEX.nLMWP⋅SOD⋅His (pEXf) ([[media:PEX.nLSH_VF_premix_fasta_6oct.txt|fasta]])
 +
 +
Sequences aligned using Geneious software, showing correct sequences for all constructs. Operon constructs all had an insertion between SpeI and PstI; this will not affect expression.
 +
 +
===Plasmid prep===
 +
''Of stored pellets and ON cultures from 5/10''
 +
 +
*E.Z.N.A. Plasmid Miniprep kit
 +
**50 μ elution volume (elution buffer)
 +
 +
{|border="1" cellpadding="1" cellspacing="0"
 +
!colspan="3"|DNA concentration
 +
|-
 +
!Sample
 +
!width="60"|Conc [ng/μl]
 +
!width="60"|A<sub>260</sub>/A<sub>280</sub>
 +
|-
 +
|pEX.nTAT&sdot;SOD&sdot;His.RBS.yCCS
 +
|align="center"|107.5
 +
|align="center"|1.96
 +
|-
 +
|pEX.nTra10&sdot;SOD&sdot;His.RBS.yCCS
 +
|align="center"|108.7
 +
|align="center"|1.93
 +
|-
 +
|pEX.nLMWP&sdot;SOD&sdot;His.RBS.yCCS
 +
|align="center"|84.35
 +
|align="center"|1.97
 +
|-
 +
|pSB1C3.His&sdot;SOD&sdot;cLMWP B
 +
|align="center"|255.4
 +
|align="center"|1.94
 +
|-
 +
|pSB1C3.His&sdot;SOD&sdot;cLMWP C
 +
|align="center"|201.2
 +
|align="center"|1.94
 +
|-
 +
|pSB1C3.His&sdot;SOD&sdot;cTAT D
 +
|align="center"|369.5
 +
|align="center"|1.92
 +
|-
 +
|pSB1C3.His&sdot;SOD&sdot;cTra10 A
 +
|align="center"|188.6
 +
|align="center"|1.95
 +
|}
 +
 +
===BL21 transformation===
 +
Quick transformation w/ 30 min on ice, 50 sec heat-shock.
 +
*30 &mu;l competent BL21(DE3)
 +
*1 &mu;l plasmid
 +
**pEX.nTra10&sdot;SOD&sdot;His.RBS.yCCS
 +
**pEX.nTAT&sdot;SOD&sdot;His.RBS.yCCS
 +
**pEX.nLMWP&sdot;SOD&sdot;His.RBS.yCCS
 +
 +
----
 +
 +
==Nina==
 +
 +
===Colony PCR on Fusion protein and protein A===
 +
 +
I screened four colonies per dish.
 +
 +
*Master mix with shipping vector verification primers (VF2 and VR):
 +
 +
[[Image:Z.jpg]]
 +
 +
*Master mix with peX verification primers:
 +
 +
[[Image:Xz.jpg]]
 +
 +
===Agarose gel===
 +
 +
I ran the PCR products on an 1 % agarose gel (110 V) in order to confirm the inserts in the desired vectors.
 +
 +
Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp
 +
 +
[[Image:Laddermixmassruler.jpg]]
 +
 +
Arrengement on the gels:
 +
 +
[[Image:Zxc.jpg|20px]]
 +
 +
*Results of the gels:
 +
 +
[[Image:As.jpg|300px]]
 +
[[Image:Ad.jpg|300px]]
 +
 +
All the Fusion and protein A bands look great, however the IgG protease bands look like they have not any insert which is bad. Tomorrow I'll run an agarose gel on my saved ligation sample of IgG protease in the gel cleaned peX vector. This will allow me to check for vector formation with a correct size and see if there has at least occured a ligation, if so I will run a new colony screen.
 +
 +
===Mini prep on His-IgG protease and His-Protein A===
 +
 +
I performed a mini prep on the inoculated colonies # 1, 2, 3 and 4 of N-terminal His-IgG protease and inoculated colonies # 1, 2, 3 and 4 of N-terminal His-Protein A.
 +
 +
 +
 +
 +
== Mimmi ==
 +
 +
=== Protein purification ===
 +
 +
{|
 +
! mix
 +
|
 +
| x2
 +
|-
 +
| NPI
 +
| 597
 +
| 1194
 +
|-
 +
| imidazole
 +
| 3
 +
| 6
 +
|-
 +
| align="right" | tot
 +
| 600µl
 +
| 1200µl
 +
|}
 +
 +
*Equilibrate column with 600µl buffer NPI-10
 +
**Centrifuge at 2900rpm for 2min
 +
 +
*Load lysate in the column (max. 600µl)
 +
**Centrifuge at 1600rpm for 5min (up to 10min)
 +
 +
{|
 +
! mix
 +
|
 +
| x7
 +
|-
 +
| NPI
 +
| 594
 +
| 4158
 +
|-
 +
| imidazole
 +
| 6
 +
| 42
 +
|-
 +
| align="right" | tot
 +
| 600µl
 +
| 4200µl
 +
|}
 +
 +
*Wash 2x (3x) with 600µl buffer NPI-20
 +
**Centrifuge at 2900rpm for 2min
 +
 +
*Elute 2x with 300µl buffer NPI-500
 +
**Centrifuge at 2900rpm for 2min
 +
**Eluate in two different tubes
 +
 +
 +
==== PhastGel ====
 +
 +
{|
 +
! well
 +
! sample
 +
| rowspan="7" | [[Image:Place_for_picture.jpg|150px|thumb|left|]]
 +
|-
 +
| 1
 +
| ladder
 +
|-
 +
| 2
 +
| SOD.his eluate 3h
 +
|-
 +
| 3
 +
| SOD.his 3h
 +
|-
 +
| 4
 +
| SOD.his 0h.
 +
|-
 +
| 5
 +
| his.SOD 3h
 +
|-
 +
| 6
 +
| his.SOD eluate 3h
 +
|}
 +
 +
==Johan==
 +
 +
===Colony PCR screen===
 +
 +
tra10-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-tat, his-bFGF-lmwp in pEX
 +
 +
5 colonies from each plate
 +
 +
0,5 µl Pol
 +
0,5 µl dNTP
 +
5 µl 5x buffer
 +
1,5 µl pex for primer
 +
1,5 µl pex rev primer
 +
16 µl H2O
 +
 +
30x mastermix
 +
 +
===Gel of PCR screen===
 +
 +
Top & bottom lanes
 +
 +
[[Image:SU 6oktgels 1.png]]
 +
 +
Top lanes
 +
 +
[[Image:SU 6oktgels.png]]
 +
 +
Compilation of all constructs. One or more bands of correct sizes for all constructs, nice!
 +
 +
 +
{{Stockholm/Footer}}

Latest revision as of 20:26, 27 October 2010

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Contents

Andreas

Sequencing results

Received sequencing results for the following samples (fasta):

  • pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 1 (VF2 & VR)
  • pSB1K3.nTra10⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
  • pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
  • pSB1K3.nTAT⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR)
  • pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 2 (VF2 & VR)
  • pSB1K3.nLMWP⋅SOD⋅His.RBS.yCCS 3 (VF2 & VR)
  • pEX.nTra10⋅SOD⋅His (pEXf) (fasta)
  • pEX.nLMWP⋅SOD⋅His (pEXf) (fasta)

Sequences aligned using Geneious software, showing correct sequences for all constructs. Operon constructs all had an insertion between SpeI and PstI; this will not affect expression.

Plasmid prep

Of stored pellets and ON cultures from 5/10

  • E.Z.N.A. Plasmid Miniprep kit
    • 50 μ elution volume (elution buffer)
DNA concentration
Sample Conc [ng/μl] A260/A280
pEX.nTAT⋅SOD⋅His.RBS.yCCS 107.5 1.96
pEX.nTra10⋅SOD⋅His.RBS.yCCS 108.7 1.93
pEX.nLMWP⋅SOD⋅His.RBS.yCCS 84.35 1.97
pSB1C3.His⋅SOD⋅cLMWP B 255.4 1.94
pSB1C3.His⋅SOD⋅cLMWP C 201.2 1.94
pSB1C3.His⋅SOD⋅cTAT D 369.5 1.92
pSB1C3.His⋅SOD⋅cTra10 A 188.6 1.95

BL21 transformation

Quick transformation w/ 30 min on ice, 50 sec heat-shock.

  • 30 μl competent BL21(DE3)
  • 1 μl plasmid
    • pEX.nTra10⋅SOD⋅His.RBS.yCCS
    • pEX.nTAT⋅SOD⋅His.RBS.yCCS
    • pEX.nLMWP⋅SOD⋅His.RBS.yCCS

Nina

Colony PCR on Fusion protein and protein A

I screened four colonies per dish.

  • Master mix with shipping vector verification primers (VF2 and VR):

Z.jpg

  • Master mix with peX verification primers:

Xz.jpg

Agarose gel

I ran the PCR products on an 1 % agarose gel (110 V) in order to confirm the inserts in the desired vectors.

Ladder: MassRuler™ DNA Ladder Mix, ready-to-use, 80-10,000 bp

Laddermixmassruler.jpg

Arrengement on the gels:

Zxc.jpg

  • Results of the gels:

As.jpg Ad.jpg

All the Fusion and protein A bands look great, however the IgG protease bands look like they have not any insert which is bad. Tomorrow I'll run an agarose gel on my saved ligation sample of IgG protease in the gel cleaned peX vector. This will allow me to check for vector formation with a correct size and see if there has at least occured a ligation, if so I will run a new colony screen.

Mini prep on His-IgG protease and His-Protein A

I performed a mini prep on the inoculated colonies # 1, 2, 3 and 4 of N-terminal His-IgG protease and inoculated colonies # 1, 2, 3 and 4 of N-terminal His-Protein A.



Mimmi

Protein purification

mix x2
NPI 597 1194
imidazole 3 6
tot 600µl 1200µl
  • Equilibrate column with 600µl buffer NPI-10
    • Centrifuge at 2900rpm for 2min
  • Load lysate in the column (max. 600µl)
    • Centrifuge at 1600rpm for 5min (up to 10min)
mix x7
NPI 594 4158
imidazole 6 42
tot 600µl 4200µl
  • Wash 2x (3x) with 600µl buffer NPI-20
    • Centrifuge at 2900rpm for 2min
  • Elute 2x with 300µl buffer NPI-500
    • Centrifuge at 2900rpm for 2min
    • Eluate in two different tubes


PhastGel

well sample
Place for picture.jpg
1 ladder
2 SOD.his eluate 3h
3 SOD.his 3h
4 SOD.his 0h.
5 his.SOD 3h
6 his.SOD eluate 3h

Johan

Colony PCR screen

tra10-bFGF-his, tat-bFGF-his, lmwp-bFGF-his, his-bFGF-tra10, his-bFGF-tat, his-bFGF-lmwp in pEX

5 colonies from each plate

0,5 µl Pol 0,5 µl dNTP 5 µl 5x buffer 1,5 µl pex for primer 1,5 µl pex rev primer 16 µl H2O

30x mastermix

Gel of PCR screen

Top & bottom lanes

SU 6oktgels 1.png

Top lanes

SU 6oktgels.png

Compilation of all constructs. One or more bands of correct sizes for all constructs, nice!






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

Retrieved from "http://2010.igem.org/Team:Stockholm/6_October_2010"