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Brown/20 June 2009
From 2009.igem.org
Team 3:
Plan for the week: • Run gels: digest OmpC, TetA: gel purify-->ligate Tet into pGEMT-easy--> DH5α transformation-->ligate OmpC-TetA standard assembly→ DH5α transformation • OmpR BB: grow cultures tonight→ genomic purification tomorrow • PCR Taz1BB • Amp-Kan-Tet plates • Mutagenic PCR
Tasks accomplished today:
1) Ashley did PCR of Taz1 -BioBrick Primers: Tar BB For (19.3 nm); EnvZ Rev (17.1 nm) -Control Primers: Amp Taz 1 For (23.0 nm); Control Taz1 Rev (26.9 nm) (i) Resuspend dry primers to 100 μM stock (ii) Make 20 μM working stock ( 1μL 100 μM stock + 4 μL H2O= 5 μL total) (iii) Template: Taz1 plasmid (miniprep) Mastermix: 47 μL Primer For + Rev: 1 μL each 3 tubes of BioBrick, 3 tubes of control PCR program: 1) 94°C for 5 min 2) 94°C for 30 sec 3) 57°C* for 30 sec 4) 72°C for 1.5 min 5) GoTo 2, 34 times 6) 72°C for 5 min 7) 4°C forever
2) Gel results for digests: 1% gel L1: 1 kb ladder L 2: Tet Steph (EcoRI, XbaI) L3: Tet MC ( E,X) L4: Tet Ash (E,X) L5: Tet Steph (E,P) L6: Tet MC (E,P) L7: Tet Ash (E,P) L8: Tet old (E,P)
<PHOTO TO GO HERE>
2% gel
L1: 1 kb L2: Omp2 MC L3: Omp1 Steph L4 : Omp 2 Steph L5: 100 bp ladder L6: 100 bp ladder
<PHOTO TO GO HERE>
• Did gel extraction on all samples
3) Making competent RU1012 (i) Plated RU1012 agar stab on LB-Kan plate (ii) 11 am: started liquid culture (iii) 6 pm: inoculate into SOB broth (2 mL, 4 mL, and 10 mL samples) (iv) check OD600 with 3 μL sample on nanodrop 11:40 am: 2 mL: 0.356 4 mL: 0.380 10 mL; 0.388 4) Testing transformation efficiency of DH5α (i) add 50 μL competent cells + 1 μL plasmid (OmpR miniprep 7a) (ii) incubate 30 mins on ice (iii) heat shock 30 sec at 42 °C (iv) incubate on ice for 2 min (v) 200 μL LB media (vi) incubate 20 min at 37°C (vii) plate different densities: 100 μL, 10 μL, 1 μL, incubate at 37°C at 4:30 pm (viii) calculate CFU
5) Gel for Taz1 Lane 1: 1 kb ladder Lane 2: 100 bp ladder Lane 3: Taz 1 Lane 4: Taz 2 Insert picture here
