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}}
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<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
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100 uL competent E.coli/reaction
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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===
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#Label 1,5 mL Eppendorf tubes and place in freezer, remove LB-Amp plates from fridge and place at room temperature.
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*Label 1,5 mL Eppendorf tubes and place in freezer, remove LB-Amp plates from fridge and place at room temperature.
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#Mix the USER cloning (<sup>TM</sup>)
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*Mix the USER<sup>TM</sup> cloning reaction in PCR tube(s).
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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"
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! Components USER mix !! x1 reaction
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|-
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| USER enzyme || 1 uL
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|-
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|  NEB buffer 4 || 0.5 uL
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|-
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|  10x BSA || 0.5 uL
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|-
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|  Backbone || 1 uL
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|-
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|}
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{| class="wikitable" style="text-align: right"
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! Components !! Negative control !! 1 fragment !! 2 fragments !! 3 fragments !! 4 fragments !! 5 fragments
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|-
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|  USER mix || 3 uL || 3 uL || 3 uL || 3 uL || 3 uL || 3 uL
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|-
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|  MQ H2O || 7 uL || - || - || - || - || -
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|-
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|  PCR product A || - || 7 uL || 3.5 uL || 2.33 uL || 1.75 uL || 1.4 uL
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|-
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|  PCR product B || - || - || 3.5 uL || 2.33 uL || 1.75 uL || 1.4 uL
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|-
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|  PCR product C || - || - || - || 2.33 uL || 1.75 uL || 1.4 uL
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|-
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|  PCR product D || - || - || - || - || 1.75 uL || 1.4 uL
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|-
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|  PCR product E || - || - || - || - || - || 1.4 uL
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|-
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|}
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*Incubate 40 minutes at 37C degrees, then 30 minutes at 25C degrees. Use old PCR machine.
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*While incubating prepare for ''E. coli'' transformation
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#label all LB-Amp plates
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#pick up competent cells from *80C freezer, thaw on ice
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#pick up 1.5 mL tubes from freezer, place on ice
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#aliquot cells, 50 uL cells per Eppendorf - pipette carefully
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#keep cells on ice
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*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)
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*Leave mixture on ice for 30 minutes (minimum 10 minutes)
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*Heat chock bacteria for 90 seconds at 42C degrees
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*leave mixture on ice for 2-5 minutes
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*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.
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*Leave at 37C degrees overnight with bottom-up
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<div style="clear: both;"></div>
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</div>
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{{: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
  1. label all LB-Amp plates
  2. pick up competent cells from *80C freezer, thaw on ice
  3. pick up 1.5 mL tubes from freezer, place on ice
  4. aliquot cells, 50 uL cells per Eppendorf - pipette carefully
  5. 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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