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Team:DTU-Denmark/Methods/USER Cloning and Transformation
From 2013.igem.org
USER Cloning and Transformation
Materials
PCR product(s) 10X diluted BSA NEB buffer 4 USER enzyme 1 PCR tube/reaction 1 Eppendorf tube/reaction 100 uL competent E.coli/reaction 1 LB-AMP plate/reaction Drigalsky's spatchula or sterile glass beads Milli-Q water 96% Ethanol Gas Burner
Procedure
- Label 1,5 mL Eppendorf tubes and place in freezer, remove LB-Amp plates from fridge and place at room temperature.
- Mix the USERTM cloning reaction in PCR tube(s).
NOTE! You may also make a control reaction, where you mix all USERTM cloning components and the backbone PCR product holding the resistance marker, but adding Milli-Q water instead of the rest of the PCR products!
| Components USER mix | x1 reaction |
|---|---|
| USER enzyme | 1 uL |
| NEB buffer 4 | 0.5 uL |
| 10x BSA | 0.5 uL |
| Backbone | 1 uL |
| Components | Negative control | 1 fragment | 2 fragments | 3 fragments | 4 fragments | 5 fragments |
|---|---|---|---|---|---|---|
| USER mix | 3 uL | 3 uL | 3 uL | 3 uL | 3 uL | 3 uL |
| MQ H2O | 7 uL | - | - | - | - | - |
| PCR product A | - | 7 uL | 3.5 uL | 2.33 uL | 1.75 uL | 1.4 uL |
| PCR product B | - | - | 3.5 uL | 2.33 uL | 1.75 uL | 1.4 uL |
| PCR product C | - | - | - | 2.33 uL | 1.75 uL | 1.4 uL |
| PCR product D | - | - | - | - | 1.75 uL | 1.4 uL |
| PCR product E | - | - | - | - | - | 1.4 uL |
- Incubate 40 minutes at 37C degrees, then 30 minutes at 25C degrees. Use old PCR machine.
- While incubating prepare for E. coli transformation
- label all LB-Amp plates
- pick up competent cells from *80C freezer, thaw on ice
- pick up 1.5 mL tubes from freezer, place on ice
- aliquot cells, 50 uL cells per Eppendorf - pipette carefully
- keep cells on ice
- After incubation is over, add the whole USER reaction to the corresponding Eppendorf with competent E. coli cells. (You may also include a negative control, where no DNA is added to the competent E.coli cells)
- Leave mixture on ice for 30 minutes (minimum 10 minutes)
- Heat chock bacteria for 90 seconds at 42C degrees
- leave mixture on ice for 2-5 minutes
- Plate bacteria using a sterile Drigalsky (sterilize with ethanol and flame between each transformation - remember to cool down the spatchula on the plate before plating out the cells). Alternatively, use sterile glass beads to spread the cells on the plate.
- Leave at 37C degrees overnight with bottom-up
