User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/29: Difference between revisions
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* Today | * Today | ||
** To create the following linkers: | ** To create the following linkers: | ||
**# SphI-PA- | **# SphI-PA-NheI linker | ||
**# SphI-PD- | **# SphI-PD-NheI linker | ||
**# SphI-PF- | **# SphI-PF-NheI linker | ||
*** [http://openwetware.org/wiki/Corum:DNA_Hybridization Hydridize] 2mM complimentary ssDNA oligonucleotides. Product is linker at 1mM concentration. Perform 1/10 serial dilutions to 100 nM final concentration. | *** [http://openwetware.org/wiki/Corum:DNA_Hybridization Hydridize] 2mM complimentary ssDNA oligonucleotides. Product is linker at 1mM concentration. Perform 1/10 serial dilutions to 100 nM final concentration. | ||
** To create the following linkers: | ** To create the following linkers: | ||
**# SphI-PB- | **# SphI-PB-NheI linker | ||
**# SphI-PG- | **# SphI-PG-NheI linker | ||
**# SphI-PL- | **# SphI-PL-NheI linker | ||
**# SphI-PLPA- | **# SphI-PLPA-NheI linker | ||
*** Perform [http://openwetware.org/wiki/Corum:PCR PCR] using ΦX174 genomic DNA as template. | *** Perform [http://openwetware.org/wiki/Corum:PCR PCR] using ΦX174 genomic DNA as template. | ||
*** Follow with [http://openwetware.org/wiki/Corum:PCR_Purification PCR purification]. Elute with 100 μL H<sub>2</sub>O and speedvac to ~20 μL. | *** Follow with [http://openwetware.org/wiki/Corum:PCR_Purification PCR purification]. Elute with 100 μL H<sub>2</sub>O and speedvac to ~20 μL. | ||
*** [http://openwetware.org/wiki/Corum:Digestion Double digest] overnight with SphI and | *** [http://openwetware.org/wiki/Corum:Digestion Double digest] overnight with SphI and NheI. | ||
* Today +1 | * Today +1 | ||
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**# SphI-PL-UTR1-NheI linker | **# SphI-PL-UTR1-NheI linker | ||
**# SphI-PLPA-UTRA-NheI | **# SphI-PLPA-UTRA-NheI | ||
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 2.5 μL pBEST-SphI//NheI-UTR1-deGFP-T500 (2nM) with 5 μL linkers (including hybridization products from yesterday) to create the following constructs: | ** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 2.5 μL pBEST-SphI//NheI-UTR1-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-NheI (including hybridization products from yesterday) to create the following constructs: | ||
**# pBEST-PA-UTR1-deGFP-T500 | **# pBEST-PA-UTR1-deGFP-T500 | ||
**# pBEST-PB-UTR1-deGFP-T500 | **# pBEST-PB-UTR1-deGFP-T500 | ||
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**# pBEST-PL-UTR1-deGFP-T500 | **# pBEST-PL-UTR1-deGFP-T500 | ||
**# pBEST-PLPA-UTR1-deGFP-T500 | **# pBEST-PLPA-UTR1-deGFP-T500 | ||
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] | ** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 0.5 μL pBEST-SphI//NcoI-deGFP-T500 (10nM) with 5 μL linkers SphI-PX-UTRX-NcoI (X = null, A, B, D, F, G, L, 'PLPA'; ~100nM) to create the following constructs: | ||
**# pBEST-null-deGFP-T500 | **# pBEST-null-deGFP-T500 | ||
**# pBEST-PA-UTRA-deGFP-T500 | **# pBEST-PA-UTRA-deGFP-T500 |
Revision as of 11:55, 29 June 2012
PHIX174 Cell Free Expression | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
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.
To do
NotesRecently Edited Notebook Pages Digital Signature
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