Team:Newcastle/16 July 2010

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*To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
*To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
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*The aim of today's experiment is to '''screen for the lacI insert'''. This could be done using PCR but that would require specific primers and is not as reliable.
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==Materials==
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*[[Team:Newcastle/15_July_2010|Overnight culture]]of transformants]]
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*Restriction enzymes ''Eco''R1 and ''Pst''1
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== Protocol ==
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==Protocol==
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#Miniprep - the [[Team:Newcastle/Qiagen_Minipreps|miniprep]] protocol was followed.
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*Plasmid extraction of transformants
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#Restriction digests - the [[Team:Newcastle/Restriction_digests|restriction digests]] protocol was followed to allow the length of the DNA to be checked using single and double digest.
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*Single (''Eco''R1) and double (''Eco''R1 and ''Pst''1) restriction digest of plasmid extracts
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#*Single digest with Pst1
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*Run digests on a gel  
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#*double digests with Pst1 and Xba1
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#Gel electrophoresis - the protocol for [[Team:Newcastle/Gel_electrophoresis|gel electrophoresis]] was followed.
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#Set up liquid broth culture in LB. The protocol for [[Team:Newcastle/Growing_an_overnight_cultures| growing an overnight culture]] was followed.
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==Results==
==Results==
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*The single digests (lanes 1-7) and the double digests (lanes 9-15) are separated by the molecular marker (lane 8).
 
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[[Image:Newcastle LacI BioBrick Construction Gel.png|700px|centre]]
[[Image:Newcastle LacI BioBrick Construction Gel.png|700px|centre]]
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''Figure 1.'' Gel electrophoresis of the single and double digests using ''Eco''R1 and ''Pst''1.
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Figure 1: Gel electrophoresis of the single and double digests using Pst1 and Xba1.
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''Lane 1-7:'' Single digests of colonies 2-8 in order
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''Lane 8:'' 1 Kbp Molecular weight marker
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''Lane 9-15:'' Double digests of colonies 2-8 in order
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This gel shows that colony 2 contains only pSB1AT3 (3 Kbp) and that the rest contain pSB1AT3 (3 Kbp) with an RFP (1.5 Kbp)insert (total 4.5 Kbp). This is as was shown in the other tests that were performed this week.
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Inference
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==Conclusion==
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We can conclude from these results that our ligation attempt has failed and that in order to carry on the construction we would need to re-perform the ligation using more of the PCR product. However this will not be done for the moment for these reasons: Firstly we believe that we can use another BioBrick already in the registry that will suit our needs, this is only made possible by the looming prospect of using Gibson cloning method to construct our Bricks; Secondly there are other Bricks that our more important for our project that we need to concentrate on.
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{{Team:Newcastle/footer}}
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{Team:Newcastle/footer}}

Revision as of 15:20, 10 August 2010

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Contents

LacI BioBrick Construction

Aims

  • To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).

Materials

Protocol

  • Plasmid extraction of transformants
  • Single (EcoR1) and double (EcoR1 and Pst1) restriction digest of plasmid extracts
  • Run digests on a gel

Results

Newcastle LacI BioBrick Construction Gel.png

Figure 1. Gel electrophoresis of the single and double digests using EcoR1 and Pst1.

Lane 1-7: Single digests of colonies 2-8 in order Lane 8: 1 Kbp Molecular weight marker Lane 9-15: Double digests of colonies 2-8 in order

This gel shows that colony 2 contains only pSB1AT3 (3 Kbp) and that the rest contain pSB1AT3 (3 Kbp) with an RFP (1.5 Kbp)insert (total 4.5 Kbp). This is as was shown in the other tests that were performed this week.

Conclusion

We can conclude from these results that our ligation attempt has failed and that in order to carry on the construction we would need to re-perform the ligation using more of the PCR product. However this will not be done for the moment for these reasons: Firstly we believe that we can use another BioBrick already in the registry that will suit our needs, this is only made possible by the looming prospect of using Gibson cloning method to construct our Bricks; Secondly there are other Bricks that our more important for our project that we need to concentrate on.

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