Team:Stockholm/2 October 2010

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(New page: {{Stockholm/Top2}} ==Andreas==)
 
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{{Stockholm/Top2}}
{{Stockholm/Top2}}
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==Andreas==
==Andreas==
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===Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX===
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''Continued from 30/8 transformations''
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====Colony PCR====
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#<u>BL21</u> pEX.nLMWP&sdot;SOD&sdot;His: A & B &dagger;
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#<u>BL21</u> pEX.nTra10&sdot;SOD&sdot;His: A & B &dagger;
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#pEX.nTAT&sdot;SOD&sdot;His.RBS.yCCS 2: A & B
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#pEX.nTAT&sdot;SOD&sdot;His.RBS.yCCS 3: A & B
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#pEX.nTra10&sdot;SOD&sdot;His.RBS.yCCS 1: A & B
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#pEX.nTra10&sdot;SOD&sdot;His.RBS.yCCS 2: A & B
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#pEX.nLMWP&sdot;SOD&sdot;His.RBS.yCCS 2: A & B
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#pEX.nLMWP&sdot;SOD&sdot;His.RBS.yCCS 3: A & B
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&dagger; ''From "Transformation of BL21, 30/8"
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Standard colony PCR settings.
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*Elongation time: 2:00
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====Gel verification====
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[[image:ColPCR_pEX.CPP_SYoperon_2oct.png|200px|thumb|right|'''Colony PCR gel verification of BL21 pEX.nCPP*SOD*His constructs and Top10 pEX.nCPP*SOD*His.RBS.yCCS operon constructs.'''<br />4 &mu;l &lambda;; 4 &mu;l sample.<br />&lambda; = O'GeneRuler 1 kb DNA ladder.]]
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0.8 % agarose, 100 V
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'''Expected bands:'''
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#744 bp
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#765 bp
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#1523 bp
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#1523 bp
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#1553 bp
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#1553 bp
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#1532 bp
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#1532 bp
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'''Results'''<br />
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Ran gel a little too long too see correct clones of constructs '''1''' and '''2'''. For the other it looks like many clones have double inserts, probably a result of too high insert:vector ratio during ligation.
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Confirmed clones:
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#-
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#-
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#A
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#B
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#-
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#-
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#B
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#B
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These will be saved for later.
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== Mimmi ==
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=== Over expression ===
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*Load PhastGel
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==== Gel ====
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{|
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! well
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! sample
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| rowspan="7" | [[Image:Place_for_picture.jpg|100px|thumb|left|]]
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! well
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! sample
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| rowspan="7" | [[Image:Place_for_picture.jpg|100px|thumb|left|]]
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|-
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| 1
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| ladder
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| 1
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| ladder
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|-
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| 2
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| SOD 0h
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| 2
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| SOD.his 3h 1:4
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|-
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| 3
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| SOD 50min
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| 3
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| SOD.his sup
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|-
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| 4
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| SOD 3h 1:4
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| 4
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| his.SOD 0h
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|-
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| 5
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| SOD sup
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| 5
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| his.SOD 3h 1:4
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|-
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| 6
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| SOD.his 0h
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| 6
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| his.SOD sup
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|}
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{{Stockholm/Footer}}

Latest revision as of 11:04, 26 October 2010

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Contents

Andreas

Transfer of nCPP⋅SOD⋅His.RBS.yCCS operon to pEX

Continued from 30/8 transformations

Colony PCR

  1. BL21 pEX.nLMWP⋅SOD⋅His: A & B †
  2. BL21 pEX.nTra10⋅SOD⋅His: A & B †
  3. pEX.nTAT⋅SOD⋅His.RBS.yCCS 2: A & B
  4. pEX.nTAT⋅SOD⋅His.RBS.yCCS 3: A & B
  5. pEX.nTra10⋅SOD⋅His.RBS.yCCS 1: A & B
  6. pEX.nTra10⋅SOD⋅His.RBS.yCCS 2: A & B
  7. pEX.nLMWP⋅SOD⋅His.RBS.yCCS 2: A & B
  8. pEX.nLMWP⋅SOD⋅His.RBS.yCCS 3: A & B

† From "Transformation of BL21, 30/8"

Standard colony PCR settings.

  • Elongation time: 2:00

Gel verification

Colony PCR gel verification of BL21 pEX.nCPP*SOD*His constructs and Top10 pEX.nCPP*SOD*His.RBS.yCCS operon constructs.
4 μl λ; 4 μl sample.
λ = O'GeneRuler 1 kb DNA ladder.

0.8 % agarose, 100 V

Expected bands:

  1. 744 bp
  2. 765 bp
  3. 1523 bp
  4. 1523 bp
  5. 1553 bp
  6. 1553 bp
  7. 1532 bp
  8. 1532 bp

Results
Ran gel a little too long too see correct clones of constructs 1 and 2. For the other it looks like many clones have double inserts, probably a result of too high insert:vector ratio during ligation.

Confirmed clones:

  1. -
  2. -
  3. A
  4. B
  5. -
  6. -
  7. B
  8. B

These will be saved for later.



Mimmi

Over expression

  • Load PhastGel

Gel

well sample
Place for picture.jpg
well sample
Place for picture.jpg
1 ladder 1 ladder
2 SOD 0h 2 SOD.his 3h 1:4
3 SOD 50min 3 SOD.his sup
4 SOD 3h 1:4 4 his.SOD 0h
5 SOD sup 5 his.SOD 3h 1:4
6 SOD.his 0h 6 his.SOD sup





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

Retrieved from "http://2010.igem.org/Team:Stockholm/2_October_2010"