Glasgow/Wetlab/Week7
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Repeated digests from 9/08 with PstI and EcoRI of minipreps thought to contain (*a→g*). There is a possibility that colony 4/1 contains the (*a→g*) insert. | Repeated digests from 9/08 with PstI and EcoRI of minipreps thought to contain (*a→g*). There is a possibility that colony 4/1 contains the (*a→g*) insert. | ||
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+ | 5 ml overnight cultures done with LB kanamycin or LB carbenicillin from the transformed cells after the site-directed mutagenesis. | ||
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Revision as of 14:41, 24 August 2007
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PROTOCOLS | REFERENCES | RESOURCES | ORDERS | BIOBRICKS UTILISED | GELS |
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.
Tuesday 14th August 2007
- Each plate of transformations of (*a→g*) and (*a→d*) grew several hundred colonies. Eight colonies from plates spread with 100ul of reaction were selected for colony PCR using Reddymix and Touch 2 program (see Protocol 9). Each selected colony was used in a reaction with primers M13_Rev_1 and *F_PstI_For_1, and then with primers M13_For_1 and *F_PstI_For_1. No expected bands were of 2kb were observed in either reaction.
- Overnights of Colonies 1 and 2 from each of the above plates (spread with 100ul of transformants) were inoculated overnight in carb LB.
Wednesday 15th August 2007
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Miniprepped overnights according to QIAGEN manual (see Protocol 5), and were labelled as follows:
Label 1/1 1/2 2/1 2/2 3/1 3/2 4/1 4/2 5/1 5/2 Plate 1 1 2 2 3 4 4 5 5 Colony 1 2 1 2 1 2 1 2 1 2 Expected Gene (*a→g*) (*a→g*) (*a→g*) (*a→g*) (*a→g*) (*a→g*) (*a→g*) (*a→g*) (*a→d*) (*a→d*) - PCR was done for each of the above minipreps with primers M13_Rev_1 and *F_PstI_For_1, and then with primers M13_For_1 and *F_PstI_For_1, used Reddymix and Touch2 (see Protocol 9). There is no evidence of a 7 gene operon insert in any of the colonies.
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Lynsey repeated amplification of XylR and XylR+Pr with KOD polymerase (see Protocol 9) using the primers listed below and subsequently cloned into TOPO vector (see Protocol 12) and transformed TOP10 cells (see Protocol 13).
- Primer Pairs:
- XylR Prefix + XylR Suffix
- Pr Prefix + XylR Suffix
- Pr_for_2 + Xylr_rev_2
- Primer Pairs:
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Maija performed PCR based site-directed mutagenesis for (*m*) to correct one PstI site and (*s*) to correct two PstI sites.
Biobrick + Construction Vector Primer Pair (*m*) + 3/20G (*m*)_SDM_PstI_1 for + (*m*)_SDM_PstI rev (*m*) + 4/6B (*s*) + 3/20G (*s*)_SDM_PstI_for + (*S*)_SDM_PstI_1 rev (*s*) + 4/6B (*s*) + 3/20G (*s*)_SDM_PstI_2 for + (*s*)_SDM_PstI_2_rev (*s*) + 4/6B KOD polymerase was used during the mutagenesis PCR, (see Protocol 9) and (seeProtocol 17)
Thursday 16th August 2007
- Repeated digests from 9/08 with PstI and EcoRI of minipreps thought to contain (*a→g*). There is a possibility that colony 4/1 contains the (*a→g*) insert.
- 5 ml overnight cultures done with LB kanamycin or LB carbenicillin from the transformed cells after the site-directed mutagenesis.
Friday 17th August 2007
- Christine did more PCR of the minipreps of the colonies suspected of containing the 7 gene operon using Reddy mix, Touch 2 and the pimer combinations below for each miniprepped colony (see Protocol 9). The result gave no evidence of the insert being present.
- M13 For and *B_EcoRI_SDM_RevI
- M13 Rev and *B_EcoRI_SDM_RevI