Glasgow/Wetlab/Week7
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To ensure successful cloning of the PCR products (*a→g*), (*a→d*) and (*d→g*) from 10/07, 1ul of GoTaq was added to each 50ul PCR reaction and extended for 10 min at 72°C. Four large lanes of (*a→g*) were run and each had a band of interest of ~8kb . One lane of each (*a→d*) and (*d→g*) was ran and only that of (*a→d*) had a band of interest of ~3kb. Each gel was 1% agarose, 50v for 40 min). | To ensure successful cloning of the PCR products (*a→g*), (*a→d*) and (*d→g*) from 10/07, 1ul of GoTaq was added to each 50ul PCR reaction and extended for 10 min at 72°C. Four large lanes of (*a→g*) were run and each had a band of interest of ~8kb . One lane of each (*a→d*) and (*d→g*) was ran and only that of (*a→d*) had a band of interest of ~3kb. Each gel was 1% agarose, 50v for 40 min). | ||
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| - | (*a→g*) and (*a→d*) were gel extracted (see [[Glasgow/Wetlab/Protocols#Protocol 11: Gel Extraction|Protocol 11]] ), cloned into TOPO vectors(see [[Glasgow/Wetlab/Protocols#Protocol 12: TOPO Cloning Reaction|Protocol 12]] ), transformed (see [[Glasgow/Wetlab/Protocols#Protocol 13: One Shot Chemical Transformation Protocol for Competent E. coli Reaction|Protocol 13]] )and grown overnight at 37°C. | + | (*a→g*) and (*a→d*) were gel extracted (see [[Glasgow/Wetlab/Protocols#Protocol 11: Gel Extraction|Protocol 11]] ), cloned into TOPO vectors(see [[Glasgow/Wetlab/Protocols#Protocol 12: TOPO Cloning Reaction|Protocol 12]] ), transformed (see [[Glasgow/Wetlab/Protocols#Protocol 13: One Shot Chemical Transformation Protocol for Competent E. coli Reaction|Protocol 13]] )and grown overnight at 37°C, each reaction divided into 100ul and ~200ul per plate. |
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Revision as of 20:56, 16 August 2007
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Contents |
Week 7
Monday 13th August 2007
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Lynsey did colony PCR (see Protocol 9) on the following E. coli TOP10 transformants to check that their plasmids carry our expected inserts:
Plasmid Expected Insert Primers pCR4 Pu Pu_Prefix_After & Pu_Suffix_After pCR4 Pu Pu_Prefix_Emma & Pu_Suffix_Emma pCR4 Pr Pr_Prefix_1 & Pr_Suffix_1 pCR4 XylR XylR_Prefix_1 & XylR_Suffix_1 pCR4 XylR & Pr Pr_Prefix_1 & XylR_Suffix_1
Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow. -
Maija did colony PCR (see Protocol 9) on the following E. coli DB3.1 transformants to check that their plasmids carry our expected inserts:
Plasmid Expected Insert Primers pSB3K5 (4/6B) (*m*) B1 17 (*m*)_for_1 & BBs_Methyl_2 pSB1AC3 (3/20G) (*m*) B1 17 (*m*)_for_1 & BBs_Methyl_2 pSB3K5 (4/6B) (*m*) C5 24 (*m*)_for_1 & (*m*)_rev_1 pSB1AC3 (3/20G) (*m*) (*m*)_for_1 & (*m*)_rev_1 pSB3K5 (4/6B) DntR DntR_Prefix_1 & DntR_Suffix_2 pSB1AC3 (3/20G) DntR DntR_Prefix_1 & DntR_Suffix_2
Overnight cultures were seeded from colonies that gave successful PCR results in order to miniprep the plasmids tomorrow.
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Maia took the following BioBricks from the kit plates and transformed (see Protocol 2) into E. coli TOP10 cells:
BioBrick Part Function Chassis Plasmid Kit Plate Location (Plate/Well) [http://partsregistry.org/Part:BBa_B0014 Bba_B0014] Double terminator E. coli TOP10 pSB1AK3 1/1G [http://partsregistry.org/Part:BBa_B0015 Bba_B0015] Double terminator E. coli TOP10 pSB1AK3 1/1I " " " " 3/3O [http://partsregistry.org/Part:BBa_J61100 Bba_J61100] RBS E. coli TOP10 pSB1A2 4/12N [http://partsregistry.org/Part:BBa_J61101 Bba_J61101] RBS E. coli TOP10 pSB1A2 4/12J [http://partsregistry.org/Part:BBa_J61102 Bba_J61102] RBS E. coli TOP10 pSB1A2 4/12L [http://partsregistry.org/Part:BBa_J61103 Bba_J61103] RBS E. coli TOP10 pSB1A2 4/12P [http://partsregistry.org/Part:BBa_E0430 Bba_E0430] EYFP + RBS + terminator E. coli TOP10 pSB1A2 1/11A [http://partsregistry.org/Part:BBa_E0422 Bba_E0422] ECFP + RBS + terminator + LVA tag E. coli TOP10 pSB1AK3 1/11G [http://partsregistry.org/Part:BBa_E0432 Bba_E0432] EYFP + RBS + terminator + LVA tag E. coli TOP10 pSB1A2 4/11C - To ensure successful cloning of the PCR products (*a→g*), (*a→d*) and (*d→g*) from 10/07, 1ul of GoTaq was added to each 50ul PCR reaction and extended for 10 min at 72°C. Four large lanes of (*a→g*) were run and each had a band of interest of ~8kb . One lane of each (*a→d*) and (*d→g*) was ran and only that of (*a→d*) had a band of interest of ~3kb. Each gel was 1% agarose, 50v for 40 min).
- (*a→g*) and (*a→d*) were gel extracted (see Protocol 11 ), cloned into TOPO vectors(see Protocol 12 ), transformed (see Protocol 13 )and grown overnight at 37°C, each reaction divided into 100ul and ~200ul per plate.
