User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/18: Difference between revisions

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==Jun6, 2012: Initial entry for PHIX174 research project==
==Jun6, 2012: Initial entry for PHIX174 research project==
===Research Log===


The current log for this research project is linked to here: [http://openwetware.org/images/6/62/PHIX174_Research_Log_Jun-18-2012.txt PHIX174_Research_Log_Jun-18-2012.txt]. It will be updated at the beginning of each week. I am currently  working on Characterization B and Hypothesis 2.
The current log for this research project is linked to here: [http://openwetware.org/images/6/62/PHIX174_Research_Log_Jun-18-2012.txt PHIX174_Research_Log_Jun-18-2012.txt]. It will be updated at the beginning of each week. I am currently  working on Characterization B and Hypothesis 2.
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Previous attempts at cloning a UTR1-deGFP linker into pBEST-pA-BamHI//XhoI-T500 backbone have failed. Sequencing showed a systematic error, whereby a truncated ~100bp piece was ligated, instead of the full UTR1-deGFP linker. Re-digesting and purifying the backbone showed the same result. Therefore, I am focusing on the linker. My hypothesis is that the PCR was mis-primed, so I designed a different set of primers to make the BamHI-UTR1-deGFP-XhoI linker. They were received today.
Previous attempts at cloning a UTR1-deGFP linker into pBEST-pA-BamHI//XhoI-T500 backbone have failed. Sequencing showed a systematic error, whereby a truncated ~100bp piece was ligated, instead of the full UTR1-deGFP linker. Re-digesting and purifying the backbone showed the same result. Therefore, I am focusing on the linker. My hypothesis is that the PCR was mis-primed, so I designed a different set of primers to make the BamHI-UTR1-deGFP-XhoI linker. They were received today.


 
* BamHI-UTR1-deGFP-XhoI-T500 sense primer: AATAATTTTGTTTAACTTTAAGAAGGAGATA 31b Th = 57.6 °C
 
** BamHI-UTR1-deGFP-XhoI-T500 sense primer: AATAATTTTGTTTAACTTTAAGAAGGAGATA 31b Th = 57.6 °C


* BamHI-UTR1-deGFP-XhoI-T500 antisense primer: ATGATAAAGAAGACAGTCATAAGTGC 26b Th = 57.3 °C
* BamHI-UTR1-deGFP-XhoI-T500 antisense primer: ATGATAAAGAAGACAGTCATAAGTGC 26b Th = 57.3 °C
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* Template: pBEST-OR2OR1Pr-UTR1-deGFP-T500
* Template: pBEST-OR2OR1Pr-UTR1-deGFP-T500


*  
* PCR product size: 861 bp (after double digestion with BamHI and XhoI, size of BamHI-UTR1-deGFP-XhoI sticky linker will be 847 bp).
 
* Using these primers and template, performed standard PCR with Th = 58 °C and an elongation time of 1 min.


* Using these primers and template, performed standard PCR with TH
===Hypothesis 3===


* Hypothesis 3: Over the weekend, I attempted whole plasmid PCR to amplify PHIX174 by whole plasmid PCR (non-mutagenic primers) by the method described in [http://nar.oxfordjournals.org/content/26/4/1126.short Chen and Ruffner]. No amplification observed. One line at 45b observed, corresponding to self-hybridization of the primers.
Over the weekend, I attempted whole plasmid PCR to amplify PHIX174 by whole plasmid PCR (non-mutagenic primers) by the method described in [http://nar.oxfordjournals.org/content/26/4/1126.short Chen and Ruffner]. No amplification observed. One line at 45b observed, corresponding to self-hybridization of the primers.


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Revision as of 14:13, 18 June 2012

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Jun6, 2012: Initial entry for PHIX174 research project

Research Log

The current log for this research project is linked to here: PHIX174_Research_Log_Jun-18-2012.txt. It will be updated at the beginning of each week. I am currently working on Characterization B and Hypothesis 2.

Characterization B

Previous attempts at cloning a UTR1-deGFP linker into pBEST-pA-BamHI//XhoI-T500 backbone have failed. Sequencing showed a systematic error, whereby a truncated ~100bp piece was ligated, instead of the full UTR1-deGFP linker. Re-digesting and purifying the backbone showed the same result. Therefore, I am focusing on the linker. My hypothesis is that the PCR was mis-primed, so I designed a different set of primers to make the BamHI-UTR1-deGFP-XhoI linker. They were received today.

  • BamHI-UTR1-deGFP-XhoI-T500 sense primer: AATAATTTTGTTTAACTTTAAGAAGGAGATA 31b Th = 57.6 °C
  • BamHI-UTR1-deGFP-XhoI-T500 antisense primer: ATGATAAAGAAGACAGTCATAAGTGC 26b Th = 57.3 °C
  • Template: pBEST-OR2OR1Pr-UTR1-deGFP-T500
  • PCR product size: 861 bp (after double digestion with BamHI and XhoI, size of BamHI-UTR1-deGFP-XhoI sticky linker will be 847 bp).
  • Using these primers and template, performed standard PCR with Th = 58 °C and an elongation time of 1 min.

Hypothesis 3

Over the weekend, I attempted whole plasmid PCR to amplify PHIX174 by whole plasmid PCR (non-mutagenic primers) by the method described in Chen and Ruffner. No amplification observed. One line at 45b observed, corresponding to self-hybridization of the primers.


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