IGEM:MIT/2005/JennyN Lab Notes

Jenny
Promoter.RBS.scFv PCR start codon to scFv 1 uL scFv-Flag Forward (with start codon) 1 uL scFv Reverse Primer 3 uL scFv 1)4-4-20, 2)S101A, 3)4M5.3 miniprepped          45 uL PCR Super Mix (-20 freezer)           ---           (3)50 uL rxns            Thermocycler: JNPCR           1. 95°C 5'           2. 95°C 1'           3. 60°C 1'           4. 68°C 3'           5. Go to 2 x29           6. 4°C forever        Test PCR on Gel           5 uL PCR reaction           5 uL H20           2 uL 6X Sample Buffer          ---          12 uL loading total each product          Use PCR product (+) and unPCRed product (-) for control.          1KB  PhiX  +4M5.3  -4M5.3  +S101A  -S101A  +4420  -4420  +FecA  -FecA       Nanodrop 4M5.3(J07014), S101A(J07013), 4420(J07012), FecA (J07019), J13002      digest Promoter.RBS BB (recipient)          5 uL 10X NEB#2          0.5 uL 100X BSA          2.5 uL SpeI          2.5 uL PstI          35 uL J13002          4.5 uL H20      digest scFvs (suffix donor) 5 uL 10X NEB#2 0.5 uL 100X BSA 2.5 uL XbaI 2.5 uL PstI 35 uL st.4M5.3, st.S101A, st.4420 4.5 uL H20 gel purify promoter.RBS, scFvs while running digest test. ligate promoter.RBS.svFv J13002.4M5.3 6 uL 4M5.3 (11.25 fm/uL) 2 uL J13002 (266.14 fm/uL) 2 uL Ligase Buffer 0.5 uL T4 DNA Ligase 9.5 uL H20 J13002.4420 3.5 uL 4420 (23.3 fm/uL) 1.5 uL J13002 2 uL buffer .5 uL Ligase 12.5 uL H20 J13002.S101A 3.5 uL S101A (18.5 fm/uL) 1.5 uL J13002 2 uL buffer .5 uL Ligase 12.5 uL H20 transform 1. Thaw cells on ice. 2. Add 2 uL rxn 3. Let incubate on ice 30' 4. Heat shock 42 for 30 sec.         5. Add 250 uL SOC or LB          6. Grow at 37 rollerdrum 1 hr.          7. Spin 1' 8. Pull off SOC/LB 9. Resuspend in 100 uL SOC/LB 10. Plate. overnights for miniprep identify correct clones (digest), streak plates overnights for freezer stock freezer stocks promoter.RBS.scFv