Team:University of Ottawa/17 July 2008

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(New page: ==Today in the Lab== '''Chris''' :'''Purification of AtCRE''' ::<li> used PCR clean up kit to purify AtCRE sample :'''Tranformation of Competent Cells''' ::<li> followed transformation of...)
 
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==Today in the Lab==
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==Today in the Lab==
'''Chris'''
'''Chris'''
:'''Purification of AtCRE'''
:'''Purification of AtCRE'''
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::<li> followed transformation of competent cells protocol to insert AtCRE plasmid into preprepared competent E. coli cells.  
::<li> followed transformation of competent cells protocol to insert AtCRE plasmid into preprepared competent E. coli cells.  
::<li> allowed cells to incubate overnight at 37 C
::<li> allowed cells to incubate overnight at 37 C
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'''Matt'''
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:'''Ligation'''
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::<li> As suggested by Dan the ligation was spiked with 1 ul ATP.
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::<li> I then performed a PCR cleanup of the Ligation product in order to purify it for transformation into competent cells.
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:'''Transformation'''
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::<li> A transformation was performed on PTP2 ligation product - I then left the streaked plates to incubate for tomorrow.
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::<li> A glycerol stock was finally made of the BY4642 yeast strain int. DQ232597.
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'''Dan'''
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:'''Gel of construct 1 after ligation'''
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::<li> Gel was unsucessful, the wrong primers were used for PCR amplification.
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::<li> Ligation seemed to work very efficiently, digesting in 50 uL with the new ClaI enzyme is working very well.
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:'''Digestions'''
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::<li> Three digestions were performed 1+T, 1+T (higher concentration of vector and insert), and just the vector
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:'''Ligations'''
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::<li>Three above plasmids were ligated overnight
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'''Tammy'''
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:'''Plasmid DNA Isolation'''
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::<li> XL10 Competent E.Coli cells transformed with pDR197::AtCKX2 were lysed and plasmid DNA was isolated using the Sigma-Aldrich kit.
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::<li> Average DNA concentration approximately 80 ng/&mu;L
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:'''Digestion of isolated pDR197::AtCKX2'''
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<TABLE BORDER CELLSPACING=2>
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<TR> <TD>'''Reaction Components'''</TD> <TD>'''1X V (&mu;L)'''</TD> </TR>
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<TR> <TD>H2O</TD> <TD>6</TD> </TR>
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<TR> <TD>Buffer 3</TD> <TD>1</TD> </TR>
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<TR> <TD>BSA</TD> <TD>1</TD> </TR>
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<TR> <TD>BamHI</TD> <TD>0.05</TD> </TR>
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<TR> <TD>PstI</TD> <TD>0.05</TD> </TR>
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<TR> <TD>DNA</TD> <TD>2</TD> </TR>
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<TR><TD>'''Total'''</TD><TD>'''10'''</TD></TR>
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</TABLE>
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<ol>
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<li> Control - No DNA (H2O)
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<li> PURE I
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<li> 1:10 II
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<li> 1:100 I
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<li> 1:100 II

Latest revision as of 23:46, 28 October 2008

Untitled Document

 

 


Contents

Today in the Lab

Chris

Purification of AtCRE
  • used PCR clean up kit to purify AtCRE sample
    • Tranformation of Competent Cells
  • followed transformation of competent cells protocol to insert AtCRE plasmid into preprepared competent E. coli cells.
  • allowed cells to incubate overnight at 37 C

    • Matt

    Ligation
  • As suggested by Dan the ligation was spiked with 1 ul ATP.
  • I then performed a PCR cleanup of the Ligation product in order to purify it for transformation into competent cells.
    • Transformation
  • A transformation was performed on PTP2 ligation product - I then left the streaked plates to incubate for tomorrow.
  • A glycerol stock was finally made of the BY4642 yeast strain int. DQ232597.

    • Dan

    Gel of construct 1 after ligation
  • Gel was unsucessful, the wrong primers were used for PCR amplification.
  • Ligation seemed to work very efficiently, digesting in 50 uL with the new ClaI enzyme is working very well.
    • Digestions
  • Three digestions were performed 1+T, 1+T (higher concentration of vector and insert), and just the vector
    • Ligations
  • Three above plasmids were ligated overnight

    • Tammy

    Plasmid DNA Isolation
  • XL10 Competent E.Coli cells transformed with pDR197::AtCKX2 were lysed and plasmid DNA was isolated using the Sigma-Aldrich kit.
  • Average DNA concentration approximately 80 ng/μL


    • Digestion of isolated pDR197::AtCKX2
    Reaction Components 1X V (μL)
    H2O 6
    Buffer 3 1
    BSA 1
    BamHI 0.05
    PstI 0.05
    DNA 2
    Total10
    1. Control - No DNA (H2O)
    2. PURE I
    3. 1:10 II
    4. 1:100 I
    5. 1:100 II
    Retrieved from "http://2008.igem.org/Team:University_of_Ottawa/17_July_2008"