diff --git a/docs/documentation/protocols.md b/docs/documentation/protocols.md index 0dec673..0c69568 100644 --- a/docs/documentation/protocols.md +++ b/docs/documentation/protocols.md @@ -48,7 +48,7 @@ Note: Try to use smaller vectors containing fewer inserts as reduced protein exp ## Gibson Assembly 1. PCR of all inserts and backbones to create appropriate overhangs -2. DpnI Digest: add 0.5 uL of DpnI to 25 uL PCR reaction, incubation 15 min at 37 °C (to remove the template DNA) +2. DpnI Digest: add 0.5 uL of DpnI to 25 uL PCR reaction, incubation 15 min at 37°C (to remove the template DNA) 3. Gel extraction of the correct PCR products: Follow the protocol of the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) 4. Mix 50–100 ng of vector DNA with a molar 2:1 or 1:1 ratio of each insert (see table below) 5. Add water and NEBuilider HiFi DNA Assembly Master Mix as indicated below @@ -60,7 +60,7 @@ Note: Try to use smaller vectors containing fewer inserts as reduced protein exp | NEBuilider HiFi DNA Assembly Master Mix | 2.5 μl |2.5 μl | | ddH2O | fill up to 5 μl |fill up to 5 μl | -6. Incubate samples in a thermocycler at 50 °C for 60 minutes +6. Incubate samples in a thermocycler at 50°C for 60 minutes 7. Store samples on ice or at –20°C for subsequent transformation @@ -72,14 +72,14 @@ To test our genetically engineered constructs, we transformed them into bacteria 2. Add 1 μl of plasmid DNA or 5 uL of assembly reaction to 20-50 μl of competent cells 3. Flick the tube to mix 4. Incubate mixture on ice for 30 minutes -5. Heat shock at the bacteria at 42 °C for 30 s +5. Heat shock at the bacteria at 42°C for 30 s 6. Place on ice for 5 minutes 7. Pipette 200 µl of room temperature LB into the mixture -8. Incubate at 37 °C for 60 minutes while shaking at 750 rpm -9. Warm selection plates to 37 °C in the incubator +8. Incubate at 37°C for 60 minutes while shaking at 750 rpm +9. Warm selection plates to 37°C in the incubator 10. Spin down bacteria and resuspend in 50 μl LB 11. Spread 50-100 μl of cells on LB agar plates containing the antibiotics for selection. When multiple antibiotics are used, use 0.6x of the standard working concentration for each antibiotic. -12. Incubate overnight at 37 °C +12. Incubate overnight at 37°C For co-transformations with two accessory plasmids: 1. Thaw competent S2060 cells on ice @@ -124,14 +124,14 @@ Day 2: Transformation 3. Add 2 µl of the ligation reaction into 50 μl of competent S2208 cells 4. Flick the tube to mix 5. Incubate mixture on ice for 30 minutes -6. Heat shock at the bacteria at 42 °C for 30 s +6. Heat shock at the bacteria at 42°C for 30 s 7. Place on ice for 5 minutes 8. Pipette 200 µl of room temperature LB into the mixture -9. Incubate at 37 °C while shaking at 750 rpm until bacteria reach saturation -10. Warm selection plates to 37 °C in the incubator +9. Incubate at 37°C while shaking at 750 rpm until bacteria reach saturation +10. Warm selection plates to 37°C in the incubator 11. Spin down bacteria and resuspend in 50 μl LB 12. Spread 50-100 μl of cells on LB agar plates -13. Incubate overnight at 37 °C +13. Incubate overnight at 37°C Day 3: Plaque Assay to determine phage titer