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Team:DTU-Denmark/Methods/USER Cloning and Transformation
From 2013.igem.org
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===Procedure=== | ===Procedure=== | ||
| - | + | *Label 1,5 mL Eppendorf tubes and place in freezer, remove LB-Amp plates from fridge and place at room temperature. | |
| - | + | *Mix the USER<sup>TM</sup> cloning reaction in PCR tube(s). | |
| + | |||
NOTE! You may also make a control reaction, where you mix all USER<sup>TM</sup> cloning components and the backbone PCR product holding the resistance marker, but adding Milli-Q water instead of the rest of the PCR products! | NOTE! You may also make a control reaction, where you mix all USER<sup>TM</sup> cloning components and the backbone PCR product holding the resistance marker, but adding Milli-Q water instead of the rest of the PCR products! | ||
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{| class="wikitable" style="text-align: right" | {| class="wikitable" style="text-align: right" | ||
! Components USER mix !! x1 reaction | ! Components USER mix !! x1 reaction | ||
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{| class="wikitable" style="text-align: right" | {| class="wikitable" style="text-align: right" | ||
! Components !! Negative control !! 1 fragment !! 2 fragments !! 3 fragments !! 4 fragments !! 5 fragments | ! Components !! Negative control !! 1 fragment !! 2 fragments !! 3 fragments !! 4 fragments !! 5 fragments | ||
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| - | + | *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 | ||
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Revision as of 13:28, 6 September 2013
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
