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)
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(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
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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
