IGEM:MIT/2005/JennyN Lab Notes

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Jenny

Promoter.RBS.scFv
   Image:Check.gif 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
Image:Check.gif 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
Image:Check.gif Nanodrop 4M5.3(J07014), S101A(J07013), 4420(J07012), FecA (J07019), J13002
Image:Check.gif 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
Image:Check.gif 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
Image:Check.gif gel purify promoter.RBS, scFvs while running digest test.
Image:Check.gif 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
Image:Check.gif 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.
Image:Pending.gif overnights for miniprep Image:Pending.gif identify correct clones (digest), streak plates Image:Pending.gif overnights for freezer stock Image:Pending.gif freezer stocks promoter.RBS.scFv
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