Glasgow/Wetlab/Week7
From 2007.igem.org
Back To Main Page | Back To Wetlab Log
Contents |
Week 7
Monday 13th August 2007
-
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.
-
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) Bba_B0014 Double terminator E. coli TOP10 pSB1AK3 1/1G Bba_B0015 Double terminator E. coli TOP10 pSB1AK3 1/1I " " " " 3/3O Bba_J61100 RBS E. coli TOP10 pSB1A2 4/12N Bba_J61101 RBS E. coli TOP10 pSB1A2 4/12J Bba_J61102 RBS E. coli TOP10 pSB1A2 4/12L Bba_J61103 RBS E. coli TOP10 pSB1A2 4/12P Bba_E0430 EYFP + RBS + terminator E. coli TOP10 pSB1A2 1/11A Bba_E0422 ECFP + RBS + terminator + LVA tag E. coli TOP10 pSB1AK3 1/11G 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
-
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 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*) - Each of the above minipreps was used for PCR 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).
-
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).
Thursday 16th August 2007
- Repeated digests from 9/08 of minipreps thought to contain (*a→g*). === Friday 17th August 2007 ===
