User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/07/09: Difference between revisions

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==Entry title==
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===Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.===


* I transformed pBEST-PA-UTR1-deGFP-T500 ligation product into JM109. Plates showed many colonies. -linker negative control showed fewer colonies, as expected. Six colonies were picked from the plate for miniculture.
, only a few colonies on the NC background, with even fewer in the backbone + linker ligation.
* Determined problem. Linker used was "SphI-PA-NcoI," when the [http://openwetware.org/images/archive/4/48/20120629160733!Promoter-UTR1-deGFP_v2.txt design file] clearly shows that the linker is "SphI-PA-NheI." So incompatible backbone was used with this linker.
* Repeated [http://openwetware.org/wiki/Corum:T4_Ligation ligation] with correct backbone (~
===Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.===
* I transformed pBEST-PA-UTRA-deGFP-T500 ligation product into JM109. Plates showed many colonies. -linker negative control showed fewer colonies, as expected. Six colonies were picked from the plate for miniculture.
===Hypothesis 2: Gene L is necessary for phage propagation.===
* Whole plasmid PCR works with the control plasmid and primers included in QuickchangeII kit.
* 5 nM ΦX174 template is functional in regular PCR.
* Therefore, the problem is with my primers. Investigating my primers...
** Sense:
   
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Revision as of 14:27, 9 July 2012

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Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.

  • I transformed pBEST-PA-UTR1-deGFP-T500 ligation product into JM109. Plates showed many colonies. -linker negative control showed fewer colonies, as expected. Six colonies were picked from the plate for miniculture.

, only a few colonies on the NC background, with even fewer in the backbone + linker ligation.

  • Determined problem. Linker used was "SphI-PA-NcoI," when the design file clearly shows that the linker is "SphI-PA-NheI." So incompatible backbone was used with this linker.
  • Repeated ligation with correct backbone (~

Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.

  • I transformed pBEST-PA-UTRA-deGFP-T500 ligation product into JM109. Plates showed many colonies. -linker negative control showed fewer colonies, as expected. Six colonies were picked from the plate for miniculture.

Hypothesis 2: Gene L is necessary for phage propagation.

  • Whole plasmid PCR works with the control plasmid and primers included in QuickchangeII kit.
  • 5 nM ΦX174 template is functional in regular PCR.
  • Therefore, the problem is with my primers. Investigating my primers...
    • Sense:
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