Team:Stockholm/7 August 2010

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* KEGG for folate: [http://www.genome.jp/kegg-bin/show_pathway?org_name=eco&mapno=00790&mapscale=&show_description=hide folate biosynthesis pathway]
* KEGG for folate: [http://www.genome.jp/kegg-bin/show_pathway?org_name=eco&mapno=00790&mapscale=&show_description=hide folate biosynthesis pathway]
* Enzyme kinetics for [http://www.genome.jp/kegg-bin/show_pathway?org_name=eco&mapno=00790&mapscale=&show_description=hide folate biosynthesis pathway] better to be extracted.
* Enzyme kinetics for [http://www.genome.jp/kegg-bin/show_pathway?org_name=eco&mapno=00790&mapscale=&show_description=hide folate biosynthesis pathway] better to be extracted.
 +
 +
----
 +
==Nina==
 +
----
 +
 +
===Tranformation of site directed mutagenesis Tyrosinase===
 +
 +
I transformed the site directed mutagenesis Tyrosinase in 100 ul Top 10 cells. The method was according to the procedure in protocols. However in step 1 I thawed the cells for 15 instead of 10 min. In step 2 I added 1 ul of DNA to the cells.
 +
 +
== Andreas ==
 +
 +
=== Cloning ===
 +
''Continued from 4/8''
 +
 +
White colonies picked from 4/8 plates.
 +
 +
{|border="1" cellpadding="2" cellspacing="0"
 +
!colspan="5"|Colony PCR samples
 +
|-
 +
|'''pSB1C3.IgG prot.'''
 +
|A (1)
 +
|B (2)
 +
|C (3)
 +
|D (4)
 +
|-
 +
|'''pSB1A3.yCCS A'''
 +
|A (5)
 +
|B (6)
 +
|C (7)
 +
|D (8)
 +
|-
 +
|'''pSB1A3.yCCS B'''
 +
|A (9)
 +
|B (10)
 +
|C (11)
 +
|D (12)
 +
|-
 +
|'''pSB1A3.SOD'''
 +
|A (13)
 +
|B (14)
 +
|C (15)
 +
|D (16)
 +
|}
 +
 +
====Colony PCR====
 +
 +
'''PCR tubes'''<br>
 +
*22.5 &mu;l dH<sub>2</sub>O
 +
*1 &mu;l VF2
 +
*1 &mu;l VR
 +
*0.5 &mu;l cell suspension
 +
*illustra Ready-to-Go PCR Beads
 +
 +
::'' '''Positive control (17):''' pSB1A3.BBa_J04450''<br>
 +
::'' '''Negative control (18):''' Blank''
 +
 +
'''PCR settings'''
 +
{|border="1" cellpadding="2" cellspacing="0"
 +
|'''1) Denaturation:'''
 +
|colspan="2"|95 &deg;C - 10:00
 +
|-
 +
|'''2) Denaturation:'''
 +
|95 &deg;C - 0:30
 +
|rowspan="3"|x30
 +
|-
 +
|'''3) Annealing:'''
 +
|55 &deg;C - 0:30
 +
|-
 +
|'''4) Elongation:'''
 +
|72 &deg;C - 1:45
 +
|-
 +
|'''5) Finish:'''
 +
|colspan="2"|25 &deg;C - &infin;
 +
|}
 +
 +
====Gel verification====
 +
 +
1 % agarose, 70 V, 1 h
 +
 +
'''Expected bands''' (plus 333 bp from vector)
 +
*'''IgG protease:''' 930 bp + 333 bp = 1263 bp<br>
 +
*'''yCCS:''' 744 bp + 333 bp = 1077 bp<br>
 +
*'''SOD:''' 459 bp + 333 bp = 792 bp<br>
 +
*'''BBa_J04450:''' 1069 bp + 317 bp = 1386 bp
 +
 +
'''Results'''<br>
 +
[[Image:ColPCR_IgGp-SOD-yCCS_7aug.png|200px|thumb|right|Colony PCR gel verification of pSB1C3.IgG prot., pSB1A3.yCCS A & B and pSB1A3.SOD.<br>
 +
'''Row 1:''' 1 kb &lambda;, 1, 2, 3, 4, 5, 6, 7, 8, 17, 1 kb &lambda;<br>
 +
'''Row 2:''' 1 kb &lambda;, 9, 10, 11, 12, 13, 14, 15, 16, 18, 1 kb &lambda;<br>
 +
3 &mu;l &lambda;, 6 &mu;l sample.]]
 +
Good bands for yCCS A & B and SOD, but not for IgG protease. The latter needs to be re-picked and amplified again.
 +
 +
Sine very strange sequencing results from yCCS A, where the sequence was that of mCherry RFP (BBa_J18932) rather than yCCS, makes me hesitant to whether samples 5-8 and 9-12 are indeed yCCS; since yCCS and mCherry nucleotide sequences are quite similar in length.<br>
 +
I will run a dedicated gel tomorrow to compare yCCS with BBa_J18932 directly.
 +
 +
====ON cultures====
 +
Clones A & B from yCCS A, yCCS B and SOD were picked for glycerol stock and plasmid prep. ON cultures were set:
 +
*5 ml LB + 100 Amp (37 &deg;C, 250 rpm)
 +
*3 ml LB + 100 Amp (30 &deg;C)
 +
 +
====Plasmid prep====
 +
Made plasmid prep of IgG protease ON cultures set 4/8. Samples were discarded since colony PCR showed that the samples were incorrect.
 +
 +
{{Stockholm/Footer}}

Latest revision as of 10:47, 26 October 2010

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Contents


Hassan

Vitamin B9 (folic acid)

  • We are trying to find a proper model for Vitamin B9 production in E. Coli
  • E. Coli lacks FolQ
  • [http://www.ncbi.nlm.nih.gov/pubmed/15611104] and [http://www.ncbi.nlm.nih.gov/pubmed/17698004] could be intereseting.
  • KEGG for folate: [http://www.genome.jp/kegg-bin/show_pathway?org_name=eco&mapno=00790&mapscale=&show_description=hide folate biosynthesis pathway]
  • Enzyme kinetics for [http://www.genome.jp/kegg-bin/show_pathway?org_name=eco&mapno=00790&mapscale=&show_description=hide folate biosynthesis pathway] better to be extracted.

Nina

Tranformation of site directed mutagenesis Tyrosinase

I transformed the site directed mutagenesis Tyrosinase in 100 ul Top 10 cells. The method was according to the procedure in protocols. However in step 1 I thawed the cells for 15 instead of 10 min. In step 2 I added 1 ul of DNA to the cells.

Andreas

Cloning

Continued from 4/8

White colonies picked from 4/8 plates.

Colony PCR samples
pSB1C3.IgG prot. A (1) B (2) C (3) D (4)
pSB1A3.yCCS A A (5) B (6) C (7) D (8)
pSB1A3.yCCS B A (9) B (10) C (11) D (12)
pSB1A3.SOD A (13) B (14) C (15) D (16)

Colony PCR

PCR tubes

  • 22.5 μl dH2O
  • 1 μl VF2
  • 1 μl VR
  • 0.5 μl cell suspension
  • illustra Ready-to-Go PCR Beads
Positive control (17): pSB1A3.BBa_J04450
Negative control (18): Blank

PCR settings

1) Denaturation: 95 °C - 10:00
2) Denaturation: 95 °C - 0:30 x30
3) Annealing: 55 °C - 0:30
4) Elongation: 72 °C - 1:45
5) Finish: 25 °C - ∞

Gel verification

1 % agarose, 70 V, 1 h

Expected bands (plus 333 bp from vector)

  • IgG protease: 930 bp + 333 bp = 1263 bp
  • yCCS: 744 bp + 333 bp = 1077 bp
  • SOD: 459 bp + 333 bp = 792 bp
  • BBa_J04450: 1069 bp + 317 bp = 1386 bp

Results

Colony PCR gel verification of pSB1C3.IgG prot., pSB1A3.yCCS A & B and pSB1A3.SOD.
Row 1: 1 kb λ, 1, 2, 3, 4, 5, 6, 7, 8, 17, 1 kb λ
Row 2: 1 kb λ, 9, 10, 11, 12, 13, 14, 15, 16, 18, 1 kb λ
3 μl λ, 6 μl sample.

Good bands for yCCS A & B and SOD, but not for IgG protease. The latter needs to be re-picked and amplified again.

Sine very strange sequencing results from yCCS A, where the sequence was that of mCherry RFP (BBa_J18932) rather than yCCS, makes me hesitant to whether samples 5-8 and 9-12 are indeed yCCS; since yCCS and mCherry nucleotide sequences are quite similar in length.
I will run a dedicated gel tomorrow to compare yCCS with BBa_J18932 directly.

ON cultures

Clones A & B from yCCS A, yCCS B and SOD were picked for glycerol stock and plasmid prep. ON cultures were set:

  • 5 ml LB + 100 Amp (37 °C, 250 rpm)
  • 3 ml LB + 100 Amp (30 °C)

Plasmid prep

Made plasmid prep of IgG protease ON cultures set 4/8. Samples were discarded since colony PCR showed that the samples were incorrect.





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

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