Team:Stockholm/28 June 2010

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= Andreas =
= Andreas =
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==Colony PCR verification of pEX vector==
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===Colony gradient PCR verification of pEX vector===
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Colony gradient PCR was run to optimize the colony PCR settings for designed pEX verification primers. Products analyzed by agarose gel electrophoresis.
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Gradient PCR was run to optimize the colony PCR settings for designed pEX verification primers (pEXf and pEXr). Products analyzed by agarose gel electrophoresis.
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===Colony PCR amplification===
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====Procedures====
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=====Materials=====
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*pEXf forward primer (CGG CTC GTA TAA TGT GTG GAA TTG)
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**Flanking insert with 116 bp
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*pEXr reverse primer (CGT TCA CCG ACA AAC AAC AG)
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**Flanking insert with 103 bp
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*PuReTaq Ready-To-Go PCR kit (GE Healthcare Life Sciences)
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=====Procedures=====
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'''PCR settings'''
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# Colony carrying pEX vector were picked from agar plate and resuspended in 10 ul LB.
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* Primers:
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#* Left to incubate in room temperature.
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**pEXf forward primer (CGG CTC GTA TAA TGT GTG GAA TTG)
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# PuReTaq Ready-To-Go PCR tubes prepared
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**pEXr reverse primer (CGT TCA CCG ACA AAC AAC AG)
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#* 1.5 ul 10uM forward primer
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* Gradient: 50°C, 55°C, 60°C
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#* 1.5 ul 10uM reverse primer
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* Elongation time: 2 min 30 s
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#* 1.0 ul template DNA (cell suspension)
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** Expected amplicon size: 116 bp (pEXf) + 103 bp (pEXr) + 1188 bp (insert) = '''1407 bp'''''
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#* 21 ul dH2O
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#* '''Total volume: 25 ul'''
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# Amplification by PCR
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#* Hot-start: 95°C - 1 min
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#* 30 cycles
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## Denaturation: 95°C - 30 s
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## Gradient (annealing): 50°C, 55°C, 60°C - 30 s
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## Elongation: 72°C - 2 min 30 s
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#* Elongation: 72°C - 10 min
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===Agarose gel electrophoresis analysis===
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'''Agarose gel analysis'''
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=====Materials=====
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*1% agarose gel
*1% agarose gel
*2 ul sample
*2 ul sample
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*1.5 ul GeneRuler 1 kb ladder (Fermentas)
 
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=====Procedures=====
 
*170 V, 20 min
*170 V, 20 min
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=====Results=====
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====Results====
'''Figure to be added later.'''
'''Figure to be added later.'''
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''Expected amplicon size: 116 bp (pEXf) + 103 bp (pEXr) + 1188 bp (insert) = '''1407 bp'''''
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=Hassan=
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May the "http://www.pathguide.org" guide us through the darkness :)
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= Mimmi =
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=== pMA ===
 +
 
 +
==== Designing primers for verification ====
 +
 
 +
*Get vector sequence from iGEM part registry
 +
 
 +
[[Image:PMA vector.jpg]]
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 +
[[Image:PMA vector linear1.jpg]]
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 +
*To be able to test in AmplifX, reajust sequence:
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 +
[[Image:PMA vector linear2.jpg]]
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*In program: Design primers
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**primer length
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**max Tm difference 2°C
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**F primer: 234-284bp
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**R primer: 634-684bp
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*Choose a primerpair with the same Tm
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*"Run PCR" -> looking good! No dimers or unspecific binding
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=== pEX ===
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==== Start over night culture for miniprep. ====
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*Mix one colony in 5ml LB containing Amp. (100µg/ml) in a falcon tube
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 +
*Leave ON in 37°C, 250rpm with some what opened lid.
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 +
{{Stockholm/Footer}}

Latest revision as of 10:35, 26 October 2010

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Contents

Andreas

Colony gradient PCR verification of pEX vector

Gradient PCR was run to optimize the colony PCR settings for designed pEX verification primers (pEXf and pEXr). Products analyzed by agarose gel electrophoresis.

Procedures

PCR settings

  • Primers:
    • pEXf forward primer (CGG CTC GTA TAA TGT GTG GAA TTG)
    • pEXr reverse primer (CGT TCA CCG ACA AAC AAC AG)
  • Gradient: 50°C, 55°C, 60°C
  • Elongation time: 2 min 30 s
    • Expected amplicon size: 116 bp (pEXf) + 103 bp (pEXr) + 1188 bp (insert) = 1407 bp

Agarose gel analysis

  • 1% agarose gel
  • 2 ul sample
  • 170 V, 20 min

Results

Figure to be added later.

Hassan

May the "http://www.pathguide.org" guide us through the darkness :)




Mimmi

pMA

Designing primers for verification

  • Get vector sequence from iGEM part registry

PMA vector.jpg

PMA vector linear1.jpg

  • To be able to test in AmplifX, reajust sequence:

PMA vector linear2.jpg

  • In program: Design primers
    • primer length
    • max Tm difference 2°C
    • F primer: 234-284bp
    • R primer: 634-684bp
  • Choose a primerpair with the same Tm
  • "Run PCR" -> looking good! No dimers or unspecific binding



pEX

Start over night culture for miniprep.

  • Mix one colony in 5ml LB containing Amp. (100µg/ml) in a falcon tube
  • Leave ON in 37°C, 250rpm with some what opened lid.





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

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