Team:DTU-Denmark/Methods/USER Cloning and Transformation

From 2013.igem.org

(Difference between revisions)
(→Procedure)
Line 18: Line 18:
#Label 1,5 mL Eppendorf tubes and place in freezer, remove LB-Amp plates from fridge and place at room temperature.
#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).
#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!
-
 
{| class="wikitable" style="text-align: right"
{| class="wikitable" style="text-align: right"
! Components USER mix !! x1 reaction
! Components USER mix !! x1 reaction
Line 33: Line 31:
|-
|-
|}
|}
-
 
{| 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
Line 52: Line 49:
|-
|-
|}
|}
-
 
#Incubate 40 minutes at 37C degrees, then 30 minutes at 25C degrees. Use old PCR machine.
#Incubate 40 minutes at 37C degrees, then 30 minutes at 25C degrees. Use old PCR machine.
#While incubating prepare for ''E.coli'' transformation
#While incubating prepare for ''E.coli'' transformation

Revision as of 13:14, 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

  1. Label 1,5 mL Eppendorf tubes and place in freezer, remove LB-Amp plates from fridge and place at room temperature.
  2. 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
  1. Incubate 40 minutes at 37C degrees, then 30 minutes at 25C degrees. Use old PCR machine.
  2. While incubating prepare for E.coli transformation

Retrieved from "http://2013.igem.org/Team:DTU-Denmark/Methods/USER_Cloning_and_Transformation"