Team:University of Ottawa/16 July 2008

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(New page: '''Innoculation of transformed X10 E.coli + pDR197::AtCKX2 in LB + AMP.''' For each dilution (PURE, 1:10, 1:100), 2 colonies were chosen to innoculate in 3 ml LM + AMP. Control is LB + AMP...)
 
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'''Innoculation of transformed X10 E.coli + pDR197::AtCKX2 in LB + AMP.''' For each dilution (PURE, 1:10, 1:100), 2 colonies were chosen to innoculate in 3 ml LM + AMP. Control is LB + AMP only.
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__TOC__
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==Today on the Lab==
 +
'''Tammy'''
 +
:'''Innoculation of transformed X10 E.coli + pDR197::AtCKX2 in LB + AMP.'''
 +
:<li>For each dilution (PURE, 1:10, 1:100), 2 colonies were chosen to innoculate in 3 ml LM + AMP.
 +
:<li>Control is LB + AMP only.
 +
:<li>Finished Innoculation at 3:oo pm
 +
'''Chris'''
 +
:'''Gel Extraction of AtCRE'''
 +
::<li> prepared a 0.8% gel and ran the AtCRE sample for 40 minutes at 90 V
 +
::<li> expected bands appeared and the desired one was excised
 +
::<li> gel extraction was performed on the excised sample successfully
 +
:'''Determining AtCRE Concentration'''
 +
::<li> measured the absorbancy of AtCRE, resulting in invalid data. It was determined that the blank was performed incorrectly, such that the machine was not zeroed properly.
 +
::<li> the absorbancy of AtCRE was remeasured using a new blank at a 1:10 dilution. The resulting absorbancy was 0.3113 at 260 nm, giving a concentration of about 150 ng/ul.
 +
:'''Ligation of AtCRE'''
 +
::<li> AtCRE was ligated with 1 ul ligase, 1 ul ATP, 2 ul DNA template, 1 uL buffer and 5 ul water
 +
::<li> the sample was incubated at 16 C overnight
 +
'''Matt'''
 +
:'''Glycerol Stock'''
 +
::<li> The BY4742 cells int 597/598 were inoculated once again for glycerol stock tomorrow - this time I sealed the tubes with the parafin.
 +
:'''Digestion'''
 +
::<li> Digestion confirmation of PTP2 with NcoI after Gel extraction was successful with correct band sizes confirming that I have the PTP2 product.
 +
::<li>A PCR cleanup was performed on the digestion product of PTP2 digested with BamHI + xhoI, concentrations were low  but not low enough that a ligation cannot be performed.
 +
::<li>I still have some pSSA42 digestion product from last time the digestion was performed that I can use for this.
 +
:'''Ligation'''
 +
::<li> A ligation was then performed using PTP2 insert in pSSA42 vector using 3:1 mol ratio - I decided to use a little more insert than suggested to ensure success of the reaction.
 +
::<li> Dan was thinking it would probably be easier if the ligation was spiked tomorrow morning with more ATP.
 +
'''Dan'''
 +
:'''Overnight PCR of 0B at 35 cycles'''
 +
::<li> Was unsucessfull
 +
:'''PCR of 0B at 35 cycles'''
 +
::<li>Was redone and did not work again,
 +
::<li> My conclusion is that the higher primer concentration is inhibiting it.

Latest revision as of 18:31, 30 July 2008

Untitled Document

 

 


Contents

Today on the Lab

Tammy

Innoculation of transformed X10 E.coli + pDR197::AtCKX2 in LB + AMP.
  • For each dilution (PURE, 1:10, 1:100), 2 colonies were chosen to innoculate in 3 ml LM + AMP.
  • Control is LB + AMP only.
  • Finished Innoculation at 3:oo pm

    • Chris

    Gel Extraction of AtCRE
  • prepared a 0.8% gel and ran the AtCRE sample for 40 minutes at 90 V
  • expected bands appeared and the desired one was excised
  • gel extraction was performed on the excised sample successfully
    • Determining AtCRE Concentration
  • measured the absorbancy of AtCRE, resulting in invalid data. It was determined that the blank was performed incorrectly, such that the machine was not zeroed properly.
  • the absorbancy of AtCRE was remeasured using a new blank at a 1:10 dilution. The resulting absorbancy was 0.3113 at 260 nm, giving a concentration of about 150 ng/ul.
    • Ligation of AtCRE
  • AtCRE was ligated with 1 ul ligase, 1 ul ATP, 2 ul DNA template, 1 uL buffer and 5 ul water
  • the sample was incubated at 16 C overnight

    • Matt

    Glycerol Stock
  • The BY4742 cells int 597/598 were inoculated once again for glycerol stock tomorrow - this time I sealed the tubes with the parafin.
    • Digestion
  • Digestion confirmation of PTP2 with NcoI after Gel extraction was successful with correct band sizes confirming that I have the PTP2 product.
  • A PCR cleanup was performed on the digestion product of PTP2 digested with BamHI + xhoI, concentrations were low but not low enough that a ligation cannot be performed.
  • I still have some pSSA42 digestion product from last time the digestion was performed that I can use for this.
    • Ligation
  • A ligation was then performed using PTP2 insert in pSSA42 vector using 3:1 mol ratio - I decided to use a little more insert than suggested to ensure success of the reaction.
  • Dan was thinking it would probably be easier if the ligation was spiked tomorrow morning with more ATP.

    • Dan

    Overnight PCR of 0B at 35 cycles
  • Was unsucessfull
    • PCR of 0B at 35 cycles
  • Was redone and did not work again,
  • My conclusion is that the higher primer concentration is inhibiting it.
    • Retrieved from "http://2008.igem.org/Team:University_of_Ottawa/16_July_2008"