User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/29: Difference between revisions
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*** 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 NcoI). | *** [http://openwetware.org/wiki/Corum:Digestion Double digest] overnight with SphI and NcoI). | ||
* Today +1 | * Today +1 | ||
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**# SphI-PL-UTR1-NcoI linker | **# SphI-PL-UTR1-NcoI linker | ||
**# SphI-PLPA-UTRA-NheI | **# SphI-PLPA-UTRA-NheI | ||
** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 0.5 μL pBEST-SphI//NcoI-UTR1-deGFP-T500 ( | ** [http://openwetware.org/wiki/Corum:T4_Ligation Ligate] 0.5 μL pBEST-SphI//NcoI-UTR1-deGFP-T500 (10nM) with 5 μL linkers (including hybridization products from yesterday) to create the following constructs: | ||
**# pBEST-PA-UTR1-deGFP-T500 | |||
**# pBEST-PB-UTR1-deGFP-T500 | **# pBEST-PB-UTR1-deGFP-T500 | ||
**# pBEST-PD-UTR1-deGFP-T500 | |||
**# pBEST-PF-UTR1-deGFP-T500 | |||
**# pBEST-PG-UTR1-deGFP-T500 | **# pBEST-PG-UTR1-deGFP-T500 | ||
**# 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] 2.5 μL pBEST-SphI//NheI-deGFP-T500 (2nM) with 5 μL linkers SphI-PX-UTRX-NheI (X = null, A, B, D, F, G, L, 'PLPA'; ~100nM) to create the following constructs: | |||
**# pBEST-null-deGFP-T500 | |||
** [http://openwetware.org/wiki/Corum: | **# pBEST-PA-UTRA-deGFP-T500 | ||
**# pBEST-PB-UTRB-deGFP-T500 | |||
**# pBEST-PD-UTRD-deGFP-T500 | |||
**# pBEST-PF-UTRF-deGFP-T500 | |||
**# pBEST-PG-UTRG-deGFP-T500 | |||
**# pBEST-PL-UTRL-deGFP-T500 | |||
** The entire list of ligation product constructs is now: | |||
**# 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-PLPA-UTRA-deGFP-T500 | **# pBEST-PLPA-UTRA-deGFP-T500 | ||
**# pBEST-NULL-deGFP-T500 | **# pBEST-NULL-deGFP-T500 | ||
* Today +2 | |||
** [http://openwetware.org/wiki/Corum:Transformation Transform] the ligation products into JM109 competent cells and grow overnight (15 plates). | |||
* Today +3 | * Today +3 | ||
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* Today +4&5 | * Today +4&5 | ||
** [http://openwetware.org/index.php?title=Corum:Miniprep Miniprep] the minicultures. Submit samples for [http://openwetware.org/wiki/Corum:DNA_Sequencing sequencing]. | ** [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). | ||
__NOTOC__ | __NOTOC__ |
Revision as of 11:36, 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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