Team:DTU-Denmark/Methods/USER Cloning and Transformation
From 2013.igem.org
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+ | {{:Team:DTU-Denmark/Templates/StartPage|USER Cloning and Transformation}} | ||
+ | <div class="overviewPage"> | ||
===Materials=== | ===Materials=== | ||
PCR product(s) | PCR product(s) | ||
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1 PCR tube/reaction | 1 PCR tube/reaction | ||
1 Eppendorf tube/reaction | 1 Eppendorf tube/reaction | ||
- | 100 uL competent E.coli/reaction | + | 100 uL competent ''E.coli''/reaction |
1 LB-AMP plate/reaction | 1 LB-AMP plate/reaction | ||
Drigalsky's spatchula or sterile glass beads | Drigalsky's spatchula or sterile glass beads | ||
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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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|- | |- | ||
|} | |} | ||
+ | *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 | ||
+ | <div style="clear: both;"></div> | ||
+ | </div> | ||
+ | {{:Team:DTU-Denmark/Templates/EndPage}} |
Latest revision as of 07:30, 30 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