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Edinburgh/30 July 2008
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* Repeated PCR for ''cenA'' and ''cex'' ('''P46''', '''P47''') from heat killed cell solution. Denaturing 95°C for 1 min, annealing 65°C for 10s, extension 70°C for 40s. Run on '''gel 30'''. Unsuccessful, perhaps because annealing temperature was too high (temperature was decided based on stock solution label, which took the prefix/suffix into consideration). (AM) | * Repeated PCR for ''cenA'' and ''cex'' ('''P46''', '''P47''') from heat killed cell solution. Denaturing 95°C for 1 min, annealing 65°C for 10s, extension 70°C for 40s. Run on '''gel 30'''. Unsuccessful, perhaps because annealing temperature was too high (temperature was decided based on stock solution label, which took the prefix/suffix into consideration). (AM) | ||
* Digested M49 and M50 (pSB1A2-rbs+''crtI'') with a) XbaI, b) SpeI/XbaI, c) sac/speI, M63 (BABEL2+rbs+''crtE'') with a) EcoRI, b) EcoRI/PstI, M67 (pSB1A2+rbs+''crtB'') with a) EcoRI/PstI, b) sacI/speI and ran on '''gel 31''' with P48 (''crtY''). (OG) | * Digested M49 and M50 (pSB1A2-rbs+''crtI'') with a) XbaI, b) SpeI/XbaI, c) sac/speI, M63 (BABEL2+rbs+''crtE'') with a) EcoRI, b) EcoRI/PstI, M67 (pSB1A2+rbs+''crtB'') with a) EcoRI/PstI, b) sacI/speI and ran on '''gel 31''' with P48 (''crtY''). (OG) | ||
| - | * Re-digested M55-M60 (pSB1A2+ | + | * Re-digested M55-M60 (pSB1A2+''P<sub>zntA</sub>'') with a) EcoRI and b) EcoRI/PstI. (This time, the amount of DNA for digestion is increased) and ran on '''gel 32'''. Results look promising for M57, but not for the others. (Yan) |
* Purified P15 (rbs+''appY'') and P28 (''crtB''), digested each with Edinbrick1 using XbaI/PstI and set-up for ligations ('''L26''' and '''L27'''). Stored overnight in the 16°C water bath. (HX)<br /> | * Purified P15 (rbs+''appY'') and P28 (''crtB''), digested each with Edinbrick1 using XbaI/PstI and set-up for ligations ('''L26''' and '''L27'''). Stored overnight in the 16°C water bath. (HX)<br /> | ||
* Ligation of pSB1A2+rbs+''dxs'' transformed to '''plates 69-70''' and ligation of ''crtE'' to BABEL2 transformed to '''plates 71-72'''. (CF) | * Ligation of pSB1A2+rbs+''dxs'' transformed to '''plates 69-70''' and ligation of ''crtE'' to BABEL2 transformed to '''plates 71-72'''. (CF) | ||
Latest revision as of 20:38, 29 October 2008
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Week 7
Wednesday 30 July 08
- P48 (crtY using a new primer mixture) and P49 (recreation of P12, rbs+dxs) created. (CF)
- Ligated putative cenA and putative cex fragments to Edinbrick 1(L28 and L29 respectively). All were digested with EcoRI/SpeI, purified and stored overnight in the 16°C water bath. (AM)
- Repeated PCR for cenA and cex (P46, P47) from heat killed cell solution. Denaturing 95°C for 1 min, annealing 65°C for 10s, extension 70°C for 40s. Run on gel 30. Unsuccessful, perhaps because annealing temperature was too high (temperature was decided based on stock solution label, which took the prefix/suffix into consideration). (AM)
- Digested M49 and M50 (pSB1A2-rbs+crtI) with a) XbaI, b) SpeI/XbaI, c) sac/speI, M63 (BABEL2+rbs+crtE) with a) EcoRI, b) EcoRI/PstI, M67 (pSB1A2+rbs+crtB) with a) EcoRI/PstI, b) sacI/speI and ran on gel 31 with P48 (crtY). (OG)
- Re-digested M55-M60 (pSB1A2+PzntA) with a) EcoRI and b) EcoRI/PstI. (This time, the amount of DNA for digestion is increased) and ran on gel 32. Results look promising for M57, but not for the others. (Yan)
- Purified P15 (rbs+appY) and P28 (crtB), digested each with Edinbrick1 using XbaI/PstI and set-up for ligations (L26 and L27). Stored overnight in the 16°C water bath. (HX)
- Ligation of pSB1A2+rbs+dxs transformed to plates 69-70 and ligation of crtE to BABEL2 transformed to plates 71-72. (CF)
- Colonies from plate 67 (pSB1A2+glgC-mut1,2) subbed to plate 73 and from plates 69-72 (pSB1A2+rbs+dxs and BABEL2+crtE) subbed to plate 74. (CF)
