Team:University of Ottawa/31 July 2008

From 2008.igem.org

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:<li> Another ligation of PTP2 insert with pSSA42 vector was performed with 3:1 ratio, 6:1 ratio, Vector with ligase, vector without ligase and H2O control.
:<li> Another ligation of PTP2 insert with pSSA42 vector was performed with 3:1 ratio, 6:1 ratio, Vector with ligase, vector without ligase and H2O control.
:<li> 2 hr ligation was unsucessful, overnight ligation was usccessful for both 3:1 and 6:1.
:<li> 2 hr ligation was unsucessful, overnight ligation was usccessful for both 3:1 and 6:1.
 +
'''Transformation'''
 +
:<li> Transformed ligation product into competent cells - 3 different plates -3:1, 6:1 and control.
 +
 +
=='''Dan'''==
 +
'''Gel Confirmation'''
 +
:<li> One gel was run for the confirmation of the ligation of constructs 0A 0B and T, this gel showed that the ligation was successful
 +
'''Gel Extraction'''
 +
:<li> The successful gel indicated that I could go forward and run all the samples on a gel for gel extraction (ran over 6 wells) and the correct bands were extracted.
 +
'''Digestion'''
 +
:<li> 2 samples were created (one for gel confirmation and one for transformation), both were digested with ClaI
 +
'''PCR cleanup'''
 +
:<li>PCR cleanup of the digestion was performed in order to prevent salts from inhibiting gel electrophoresis.
 +
'''Ligation'''
 +
:<li> Ligation was run overnight in the thermocycler of pSSA140B and p1T(0A0B)

Latest revision as of 15:05, 12 August 2008

Untitled Document

 

 

Contents

Today in the Lab

Chris

Microwave Digestion Experiment

  • Following protocol outlined online, tested a microwave protocol to hasten digestion period.
  • Digested DQ232597 with HindIII. Ran five samples: 5 seconds in micro, 10 seconds, 15 seconds, 10 seconds in and 2 minutes out (four times), water control, water control 10 seconds in microwave and 2 minutes out, 1 hr traditional digest.
  • Denatured HindIII at 80 C for 20 minutes, then ran products on a gel.
  • Results promising; however, more experimentation needed to confirm validity of this protocol

    • Matt

    Ligation

  • Another ligation of PTP2 insert with pSSA42 vector was performed with 3:1 ratio, 6:1 ratio, Vector with ligase, vector without ligase and H2O control.
  • 2 hr ligation was unsucessful, overnight ligation was usccessful for both 3:1 and 6:1.

    • Transformation

  • Transformed ligation product into competent cells - 3 different plates -3:1, 6:1 and control.

    • Dan

    Gel Confirmation

  • One gel was run for the confirmation of the ligation of constructs 0A 0B and T, this gel showed that the ligation was successful

    • Gel Extraction

  • The successful gel indicated that I could go forward and run all the samples on a gel for gel extraction (ran over 6 wells) and the correct bands were extracted.

    • Digestion

  • 2 samples were created (one for gel confirmation and one for transformation), both were digested with ClaI

    • PCR cleanup

  • PCR cleanup of the digestion was performed in order to prevent salts from inhibiting gel electrophoresis.

    • Ligation

  • Ligation was run overnight in the thermocycler of pSSA140B and p1T(0A0B)
    • Retrieved from "http://2008.igem.org/Team:University_of_Ottawa/31_July_2008"