Team:Hawaii/Construction of pRL1383a

From 2008.igem.org

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== Construction of pRL1383a ==
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==Ligation of Parts==
* The BioBrick parts of pRL1383a are to be ligated in a series of experiments.  
* The BioBrick parts of pRL1383a are to be ligated in a series of experiments.  
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==Parts==
 
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{| border="1"
 
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|+ '''Units of pRL1383a Construct'''
 
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!width="100"|Name
 
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!width="300"|Status
 
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!width="300"|Quantity
 
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!width="300"|Individual construct
 
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|-
 
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|OriV
 
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|PCR amplified 7/30
 
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|?
 
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|pSB1A3::OriV
 
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|-
 
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|rep region
 
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|PCR amplified [[Team:Hawaii/Notebook/2008-07-29|7/29]]
 
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|~960ng
 
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|P<sub>lac</sub>-RBS-rep
 
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|-
 
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|omega interposon
 
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|PCR amplified [[Team:Hawaii/Notebook/2008-07-30|7/30]]
 
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|?
 
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|pSB1A3::OriV
 
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|-
 
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|aadA gene
 
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|PCR amplified [[Team:Hawaii/Notebook/2008-07-29|7/29]]
 
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|128ng
 
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|P<sub>lac</sub>-RBS-aadA-TT
 
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|-
 
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|oriT
 
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|Verified in vector [[Team:Hawaii/Notebook/2008-07-30|7/30]]
 
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|0(need plasmid prep, glycerol stock)
 
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|pSB1A3::OriT
 
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|-
 
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|P1 lytic replication origin
 
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|Primers ordered 7/30
 
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|n/a
 
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|pSB1A3::P1 lytic rep origin
 
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|-
 
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|P<sub>lac</sub>[http://partsregistry.org/Part:BBa_I14032 BBa_I14032]
 
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|verified by colony PCR
 
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|need glycerol stock and plasmid prep
 
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|P<sub>lac</sub>-RBS-aadA-TT, P<sub>lac</sub>-RBS-rep
 
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|-
 
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|RBS, efficiency 1.0[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]
 
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|plasmid available
 
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|? need to check
 
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|P<sub>lac</sub>-RBS-aadA-TT, P<sub>lac</sub>-RBS-rep
 
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|-
 
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|Terminator [http://partsregistry.org/Part:BBa_B0015 BBa_B0015]
 
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|plasmid prep [[Team:Hawaii/Notebook/2008-07-30|7/30]]
 
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|needs quantification
 
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|P<sub>lac</sub>-RBS-aadA-TT
 
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|-
 
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|BioBrick Base Vector[http://partsregistry.org/Part:BBa_I51020 BBa_I51020]
 
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|plasmid prep [[Team:Hawaii/Notebook/2008-07-30|7/30]]
 
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|needs quantification
 
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|all parts will be inserted
 
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|}
 
== Methods ==
== Methods ==

Latest revision as of 02:36, 6 August 2008

Projects Events Resources
Sponsors Experiments Milestones Protocols
Notebook (t) Meetings (t)

Contents

Ligation of Parts

  • The BioBrick parts of pRL1383a are to be ligated in a series of experiments.


Methods

  1. Round 1 of Restriction Digest and Ligation
Restriction Digest & Ligation: Round 1
Name Digest 1 Digest 2 Ligation to Product
mob XbaI & PstI n/a [http://partsregistry.org/Part:BBa_B0024 BBa_B0024] TT-mob
rep XbaI & PstI n/a [http://partsregistry.org/Part:BBa_R0010 BBa_R0010] Plac-rep
oriV XbaI & PstI n/a [http://partsregistry.org/Part:pSB1A3 pSB1A3] pSB1A3::oriV
aadA region XbaI & PstI n/a [http://partsregistry.org/Part:BBa_R0010 BBa_R0010] Plac-aadA
oriT XbaI & PstI n/a [http://partsregistry.org/Part:pSB1A3 pSB1A3] pSB1A3::oriT
pSB1A3 XbaI & PstI n/a see oriT and oriV see oriT and oriV
[http://partsregistry.org/Part:BBa_B0024 BBa_B0024] SpeI PstI see mob region above see mob region above
[http://partsregistry.org/Part:BBa_R0010 BBa_R0010] SpeI PstI see rep and aadA region above see rep and aadA region above
  1. In gel purification of ligation products
  1. Round 2 of Restriction Digest and Ligation
Restriction Digest & Ligation: Round 2
Name Digest 1 Digest 2 Ligation to Product
TT-mob EcoRI SpeI [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] TT-mob-TT
Plac-aadA EcoRI SpeI [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] Plac-aadA -TT
[http://partsregistry.org/Part:BBa_B0015 BBa_B0015] EcoRI XbaI see mob and aadA above see mob and aadA above
  1. Transformation
  1. Verification with Colony PCR

Results


Discussion

  • What was learned and how to do future experiments differently.


[http://manoa.hawaii.edu/ Sponsor_UHM.gif][http://manoa.hawaii.edu/ovcrge/ Sponsor_OVCRGE.gif][http://www.ctahr.hawaii.edu Sponsor_CTAHR.gif]

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