Team:Stockholm/10 August 2010

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=== Gels ===
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==== MITF ====
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::'''verification'''
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*Nothing...!
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==== yCCS and RFP ====
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::'''verification'''
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*See Andreas notes
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==== yCCS and SOD ====
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::'''site-directed'''
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[[Image:2010-08-10_yCCS_+_SOD_site_directed_mutagsenesis.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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| yCCS
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|-
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| 3
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| blank
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|-
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| 4
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| plasmid control
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|-
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| 5
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| ladder
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|-
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| 6
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| SOD
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|-
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| 7
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| blank
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|-
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| 8
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| plasmid control
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|}
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----
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==Nina==
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=== Mini prep of Tyrosinase===
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I performed a mini prep on the six samples of falcon tubes containing inoculated tyrosinase in its original vector that have been site directed mutagenesis. The colonies are: #2, 3, 4, 5, 6, ,7 & 8. The method was according to the procedure described under Protocols.
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Spectophotometer:
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[[Image:Tab.jpg]]
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----
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===Measuring concentration of protein A (ZZ domain) and IgG protease in bank vector C===
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I measured the complately digested protein A ZZ domain (PCR product) and the bank vector C that before the digestion contained the IgG protease gene.
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Spectophotometer:
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[[Image:Tabn.jpg]]
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 +
----
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===Ligation of protein A (ZZ domain) with bank vector C===
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 +
I ligated protein A ZZ domian into the digested bank vector C to be able to make fusion proteins with IgG protease and CPP but also to send in this part to iGEM hq since they want the genes to be sent in this vector.
 +
 +
The vector concentraion should preferably be 25 ng/ul when performing ligation with a gene.
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 +
I used:
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*bank vector C 0.5 ul
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*protein A gene 3 ul
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*quick ligase 1 ul
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*quicke ligase buffer 2X 3 ul
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Incubated in RT 15 min
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----
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===Transformation of protein A (ZZ domain) in bank vector C===
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I transformed the ligated protein A (ZZ domain) in bank vector C in 100 ul Top 10 cells. 
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 +
The transformation method is according to the procedure decribed in protocols. However in the step 1 I thawed for 15 min instead of 10 min. In step 2 I added 3 ul of DNA, in step 6 I used LB instead of SOC and finally in step 10 I spread 50 ul of sample on a chloramphenicol plate.
==Andreas==
==Andreas==
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Plates incubated ON in 37 °C.
Plates incubated ON in 37 °C.
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{{Stockholm/Footer}}

Latest revision as of 10:49, 26 October 2010

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↳   Notebook Safety Protocols



Contents

Mimmi

yCCS and SOD

redoing the Site-Directed Mutagenesis

Mix (µl) X2 X2 yCCS A SOD A conditions
sH2O 40 80 yCCS_mut_F SOD_mut_F time °C
dNTPs 1 2 yCCS_mut_R SOD_mut_R 30s 95
F primer 1 2 pSB1C3 pSB1C3 30s 95 )
R primer 1 2 ~3.5kb ~3.2kb 30s 55 > 22 cycles
DNA 1 2X1 4m 68 )
Pfu buffer 5 10 oo 10
Pfu turbo 1 2
tot 50
SOD Primers
SOD_mut_F 262.55µl H2O --> 100µM
SOD_mut_R 372.64µl H2O --> 100µM
SOD_mut_F 3µl + 24.15µl H2O --> 125ng/µl
SOD_mut_R 3µl + 24.42µl H2O --> 125ng/µl


MITF

Amplifying

Mix phusion (µl) /4 Mix Pfu turbo (µl) /4 Primers
sH2O 67 sH2O 77 MITF_F
F primer 5 F primer 5 MITF_R
R primer 5 R primer 5
dNTP 2 dNTP 2 conditions
5X buffer 20 10X buffer 10 time °C
Phusion pol. 1 Pfu pol. 1 2m 98
tot 100 25 tot 100 25 30s 98 )
30s 40-55 > 5 cycles
1m30s 72 )
30s 98 )
30s 65 > 25 cycles
1m30s 72 )
10m 72
oo 10


Gels

MITF

verification
  • Nothing...!


yCCS and RFP

verification
  • See Andreas notes


yCCS and SOD

site-directed
2010-08-10 yCCS + SOD site directed mutagsenesis.jpg
well sample
1 ladder
2 yCCS
3 blank
4 plasmid control
5 ladder
6 SOD
7 blank
8 plasmid control


Nina

Mini prep of Tyrosinase

I performed a mini prep on the six samples of falcon tubes containing inoculated tyrosinase in its original vector that have been site directed mutagenesis. The colonies are: #2, 3, 4, 5, 6, ,7 & 8. The method was according to the procedure described under Protocols.

Spectophotometer:

Tab.jpg

Measuring concentration of protein A (ZZ domain) and IgG protease in bank vector C

I measured the complately digested protein A ZZ domain (PCR product) and the bank vector C that before the digestion contained the IgG protease gene.

Spectophotometer:

Tabn.jpg

Ligation of protein A (ZZ domain) with bank vector C

I ligated protein A ZZ domian into the digested bank vector C to be able to make fusion proteins with IgG protease and CPP but also to send in this part to iGEM hq since they want the genes to be sent in this vector.

The vector concentraion should preferably be 25 ng/ul when performing ligation with a gene.

I used:

  • bank vector C 0.5 ul
  • protein A gene 3 ul
  • quick ligase 1 ul
  • quicke ligase buffer 2X 3 ul

Incubated in RT 15 min

Transformation of protein A (ZZ domain) in bank vector C

I transformed the ligated protein A (ZZ domain) in bank vector C in 100 ul Top 10 cells.

The transformation method is according to the procedure decribed in protocols. However in the step 1 I thawed for 15 min instead of 10 min. In step 2 I added 3 ul of DNA, in step 6 I used LB instead of SOC and finally in step 10 I spread 50 ul of sample on a chloramphenicol plate.

Andreas

Cloning of IgG protease

Continued from 9/8 ON plates

Colony PCR

Four clones (A, B, C, D) were picked for colony PCR. Positive control: pSB1C3.BBa_J04450 plasmid. Procedures according to protocol; 1:40 elongation time.

Gel verification

1 % agarose, 90 V, 40 min

Results: Weak and very irregularly sized bands. Probably something wrong with the ligation mixture. New IgGp digestion will be made.

New IgG protease cloning

Digestion

Used pSB1A3.IgGp at a conc. of 139 ng/μl.

10X FD buffer 2 μl
dH2O 3 μl
2 μg DNA 14 μl
FD SpeI 0.5 μl
FD EcoRI 0.5 μl
  20 μl

Incubation: 37 °C, 30 min

Ligation

Used pre-digested (EcoRI & SpeI) pSB1C3 vector from 3/8.

pSB1C3 vector 2.5 μl
IgGp insert 12.5 μl †
5X rapid ligation buffer 4 μl
T4 DNA ligase 1 μl

† Since I used the ligation protocol from 3/8, I mistakenly calculated that 12.5:2.5 would give a 5:1 insert:vector ratio. However, due to the new digestion volume, this actually led to a 12.5:1 ratio.

Transformation

Transformation into 100 μl comptetent Top10.

  1. Quick-transformation
    1. 3 μl ligation mix. Incubation 5 min on ice.
    2. Heat-shock 30 sec in 42 °C. Cells cooled on ice.
    3. 100 μl plated onto Cm 25 plate.
  2. Standard transformation
    • 3 μl ligation mix.
    • Standard protocol transformation procedures.

Plates incubated ON in 37 °C.





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

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