User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/28: Difference between revisions
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* Characterization C showed no colonies after transformation. Therefore, I decided to characterize all linkers and backbones for those constructs as well as the UTR1-deGFP constructs via [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification]. [http://openwetware.org/wiki/Image:Quanitfluore_062812.txt Results] were as follows: | * Characterization C showed no colonies after transformation. Therefore, I decided to characterize all linkers and backbones for those constructs as well as the UTR1-deGFP constructs via [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification]. [http://openwetware.org/wiki/Image:Quanitfluore_062812.txt Results] were as follows: | ||
** SphI-PA- | ** SphI-PA-NheI linker = 23 nM | ||
** SphI-PB- | ** SphI-PB-NheI linker = 7 nM | ||
** SphI-PD- | ** SphI-PD-NheI linker = 18 nM | ||
** SphI-PF- | ** SphI-PF-NheI linker = 24 nM | ||
** SphI-PG- | ** SphI-PG-NheI linker = 10 nM | ||
** SphI-PL- | ** SphI-PL-NheI linker = 11 nM | ||
** SphI-PLPA- | ** SphI-PLPA-NheI linker = 4 nM | ||
** pBEST-SphI// | ** pBEST-SphI//NheI-UTR1-deGFP-T500 backbone = 2 nM | ||
* | * * The backbone is at expected 2 nM concentration but is needed at ~10 nM concentration, so to obtain a proper ligation with 1:100 backbone to linker concentration, the volume of backbone needs to be increased by a factor of 5 in ligation, i.e., 2.5 μL backbone and 5 μL reconstucted linkers. | ||
* The backbone is at expected ~10 nM concentration, so I kept it for use in the next ligation. | * The backbone is at expected ~10 nM concentration, so I kept it for use in the next ligation. | ||
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* There were no colonies after transformation. Therefore, I decided to characterize all linkers and backbones for those constructs as well as the UTR1-deGFP constructs via [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification]. [http://openwetware.org/wiki/Image:Quanitfluore_062812.txt Results] were as follows: | * There were no colonies after transformation. Therefore, I decided to characterize all linkers and backbones for those constructs as well as the UTR1-deGFP constructs via [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification]. [http://openwetware.org/wiki/Image:Quanitfluore_062812.txt Results] were as follows: | ||
** SphI-null- | ** SphI-null-NcoI linker = 73 nM | ||
** SphI-PA-UTRA- | ** SphI-PA-UTRA-NcoI linker = 46 nM | ||
** SphI-PB-UTRB- | ** SphI-PB-UTRB-NcoI linker = 37 nM | ||
** SphI-PD-UTRD- | ** SphI-PD-UTRD-NcoI linker = 66 nM | ||
** SphI-PF-UTRF- | ** SphI-PF-UTRF-NcoI linker = 66 nM | ||
** SphI-PG-UTRG- | ** SphI-PG-UTRG-NcoI linker = 139 nM | ||
** SphI-PL-UTRL- | ** SphI-PL-UTRL-NcoI linker = 87 nM | ||
** SphI-PLPA-UTRA- | ** SphI-PLPA-UTRA-NcoI raw PCR product = 166 nM | ||
** pBEST-SphI// | ** pBEST-SphI//NcoI-deGFP-T500 backbone = 10 nM | ||
* All of the linkers are ~100 nM, which is what is expected for proper ligation. | * All of the linkers are ~100 nM, which is what is expected for proper ligation. | ||
* The backbone is at expected | * The backbone is at expected 10 nM concentration, so I performed [http://openwetware.org/wiki/Corum:T4_Ligation ligation] with 0.5 μL backbone and 5 μL linkers to obtain the proper 1:100 ratio, respectively. | ||
** I digested and then [http://openwetware.org/index.php?title=Corum:PCR_Purification PCR purified] the SphI-PLPA-UTRA-NheI raw PCR product with SphI and NheI | ** I accidentally digested and then [http://openwetware.org/index.php?title=Corum:PCR_Purification PCR purified] the SphI-PLPA-UTRA-NheI raw PCR product with SphI and NheI, so this PCR will have to be repeated, since I needed to have digested with *NcoI*, NOT NheI. | ||
===Hypothesis 2: Gene L is necessary for phage propagation.=== | ===Hypothesis 2: Gene L is necessary for phage propagation.=== |
Revision as of 09:13, 29 June 2012
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Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.
Characterization C: Expression of PHIX174 promoters fused to UTRX-deGFP.
Hypothesis 2: Gene L is necessary for phage propagation.
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