User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/29: Difference between revisions
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==To do== | ==To do== | ||
* Today | * Today | ||
** To create the following linkers: | |||
**# SphI-PA-NcoI linker | |||
**# SphI-PD-NcoI linker | |||
**# SphI-PF-NcoI 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. | |||
** To initiate the following linkers: | |||
**# SphI-PB-NcoI linker | |||
**# SphI-PG-NcoI linker | |||
**# SphI-PL-NcoI linker | |||
**# SphI-PLPA-NcoI linker | |||
*** 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. | |||
*** [http://openwetware.org/wiki/Corum:Digestion Double digest] overnight with SphI and NcoI. | |||
* Today +1 | |||
** To obtain following linkers, perform [http://openwetware.org/wiki/Corum:Gel_Purification Gel extraction] (elute in 100 μL H<sub>2</sub>O, linkers should be at ~100nM concentration): | |||
**# SphI-PB-NcoI linker | |||
**# SphI-PG-NcoI linker | |||
**# SphI-PL-NcoI linker | |||
**# SphI-PLPA-NcoI linker | |||
** To create the following linkers: | ** To create the following linkers: | ||
**# SphI-PA-NheI linker | **# SphI-PA-NheI linker | ||
**# SphI-PB-NheI linker | |||
**# SphI-PD-NheI linker | **# SphI-PD-NheI linker | ||
**# SphI-PF-NheI linker | **# SphI-PF-NheI linker | ||
**# SphI-PG-NheI linker | **# SphI-PG-NheI linker | ||
**# SphI-PL-NheI linker | **# SphI-PL-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. | |||
** To initiate the following linkers: | |||
**# SphI-PLPA-NheI linker | **# 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 NcoI. | ||
* Today + | *Today +3 | ||
** To obtain following linkers, perform [http://openwetware.org/wiki/Corum:Gel_Purification Gel extraction] (elute in 100 μL H<sub>2</sub>O, linkers should be at ~100nM concentration): | ** To obtain following linkers, perform [http://openwetware.org/wiki/Corum:Gel_Purification Gel extraction] (elute in 100 μL H<sub>2</sub>O, linkers should be at ~100nM concentration): | ||
**# SphI-PB- | **# SphI-PLPA-NheI linker | ||
**# SphI-PG- | ** The entire list of linkers is now as follows: | ||
**# SphI-PL- | **# SphI-PA-NcoI | ||
**# SphI-PB-NcoI | |||
**# SphI-PD-NcoI | |||
**# SphI-PF-NcoI | |||
**# SphI-PG-NcoI | |||
**# SphI-PL-NcoI | |||
**# SphI-PLPA-NcoI | |||
**# SphI-PA-UTRA-NheI | |||
**# SphI-PB-UTRB-NheI | |||
**# SphI-PD-UTRD-NheI | |||
**# SphI-PF-UTRF-NheI | |||
**# SphI-PG-UTRG-NheI | |||
**# SphI-PL-UTRL-NheI | |||
**# SphI-PLPA-UTRA-NheI | **# SphI-PLPA-UTRA-NheI | ||
** [http://openwetware.org/wiki/Corum: | **# SphI-NULL-NheI | ||
**# pBEST- | ** Verify that linker concentrations are all ~100nM with [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification]. | ||
** The list of backbones is: | |||
**# pBEST-SphI//NcoI-UTR1-deGFP-T500 (10nM). [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 0.5 μL with 5 μL SphI-PX-NcoI linkers. | |||
**# pBEST-SphI//NheI-deGFP-T500 (2nM) [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 0.5 μL with 5 μL SphI-PX-UTRX-NheI linkers. | |||
** The entire list of ligation product constructs is now: | ** The entire list of ligation product constructs is now: | ||
**# pBEST-PA-UTR1-deGFP-T500 | **# pBEST-PA-UTR1-deGFP-T500 | ||
Line 73: | Line 94: | ||
**# pBEST-NULL-deGFP-T500 | **# pBEST-NULL-deGFP-T500 | ||
* Today + | * Today +4 | ||
** [http://openwetware.org/wiki/Corum:Transformation Transform] the ligation products into JM109 competent cells and grow overnight (15 plates). | ** [http://openwetware.org/wiki/Corum:Transformation Transform] the ligation products into JM109 competent cells and grow overnight (15 plates). | ||
* Today + | * Today +5 | ||
** Select 4 colonies per plate and grow 2.5 mL miniculures (4 × 15 = 60 total). | ** Select 4 colonies per plate and grow 2.5 mL miniculures (4 × 15 = 60 total). | ||
* Today + | * Today +6&7 | ||
** [http://openwetware.org/index.php?title=Corum:Miniprep Miniprep] the minicultures. Submit samples for [http://openwetware.org/wiki/Corum:DNA_Sequencing sequencing] ((4 × 15 = 60 total). | ** [http://openwetware.org/index.php?title=Corum:Miniprep Miniprep] the minicultures. Submit samples for [http://openwetware.org/wiki/Corum:DNA_Sequencing sequencing] ((4 × 15 = 60 total). | ||
Revision as of 13:06, 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
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