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(New page: {{Template:UIUC_Illinois_Bioware}} {{Template:UIUC_Illinois_Bioware_Background}} == Protocols == <h3>Making Electro-Competent Cells</h3> <html> Materials:<br> • DH5alpha glycerol stock...) |
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== Protocols == | == Protocols == | ||
| - | <h3>Making | + | <h3>Making Electrocompetent Cells</h3> |
| - | + | SOB | |
| - | + | 2% tryptone | |
| - | + | 0.5% yeast extract | |
| - | + | 10 mM NaCl | |
| - | + | 2.5 mM KCl | |
| - | + | 10 mM MgCl2 | |
| - | + | 10 mM MgSO4 | |
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| + | SOC | ||
| + | SOB + 20 mM glucose | ||
| + | Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol). | ||
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| + | Procedure (for 2, 250 ml cultures) | ||
| + | 1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm. | ||
| + | 2. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks. | ||
| + | 3. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells | ||
| + | 4. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles. | ||
| + | 5. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth. | ||
| + | 6. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down. | ||
| + | 7. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells as before. | ||
| + | 8. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. | ||
| + | 9. At this point you can electroporate or freeze the cells away. To freeze, Add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen transfer them to a -80°C freezer. The cultures should be good for >6 months. | ||
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<h3>Site Directed Mutagenesis</h3> | <h3>Site Directed Mutagenesis</h3> | ||
Revision as of 01:26, 27 October 2010
Protocols
Making Electrocompetent Cells
SOB 2% tryptone 0.5% yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4
SOC SOB + 20 mM glucose Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol).
Procedure (for 2, 250 ml cultures) 1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37oC and 250 rpm. 2. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks. 3. Shake at 37°C and 250 rpm until the cultures reach an OD600 of 0.5-0.7. Be sure to turn on centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells 4. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles. 5. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth. 6. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down. 7. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells as before. 8. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. 9. At this point you can electroporate or freeze the cells away. To freeze, Add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the culture transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen transfer them to a -80°C freezer. The cultures should be good for >6 months.
Site Directed Mutagenesis
Procedure modified from Stratagene QuikChange II Protocol
Materials
- PFU Ultra Polymerase (high fidelity)
- 10X Reaction Buffer
- DpnI (20U/μL)
- dNTPs
- Competent Cells
1. Design primers
2. Mutant strand synthesis (PCR)
3. DpnI digestion of template
4. Transform and plate
Primer Design Considerations
• Both primers must contain desired mutation and anneal to same sequence on opposite strands of the plasmid.
• Keep the Primers aruond 25-45 bp long with 10-15 bp on either side of the mutated base.
• Keep the melting temperature greater than or equal to 78°C
Tm=81.5+,41(%GC)-675/N-%mismatch where N is the length of the primer
• Try to keep the primers to a minimum GC content of 40% and end the primer in GC for a clamp.
• Make sure primers are in excess to template
PCR
| Ingredient | Amount |
| 10X Buffer | 5 μL |
| Template | 20 ng |
| Primer 1 | 125 ng |
| Primer 2 | 125 ng |
| 10 mM dNTPs | 1 μL |
| Polymerase | 1 μL |
| H2O | Bring to 50 μL |
| 95°C | 3 min |
| 95°C | 30 sec |
| annealing temp | 1 min |
| 72°C | 1 min/Kb |
| go to step 2 | 12X |
| 72°C | 1 min | hold at 4°C |
DpnI Digestion
| DNA | 500 ng |
| Buffer 4 | 5 μL |
| BSA | .5 μL |
| DpnI | 1 μL |
| H2O | Bring to 50 μL |
Transform via electroporation
