Brown: Team Resistance/3 July 2008

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# pDKL02 + IPTG - growth
# pDKL02 + IPTG - growth
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*These results indicate that the pDKL02 promoter is rather leaky and not as tight as the Arabinose induced promoter.  The clear solution of pVJ4 and Arabinose was placed in more LB to see if growth would occur.  There was no further growth signifying that cell lysis had occurred.  The R gene of the cassette dissolves the membrane when expressed hence the clearing of the solution.
+
* The clear solution of pVJ4 and Arabinose was placed in more LB to see if growth would occur.  There was no further growth signifying that cell lysis had occurred.  The R gene of the cassette degrades the membrane when expressed hence the clearing of the solution.
 +
 
 +
*The IPTG solution did not contain lactose, which is probably the reason it did not work
 +
 
 +
 
 +
OD measurements after pVJ4 cultures were left over night
 +
+IPTG: 0.084 (compare 0.070 last night)
 +
+arabinose: 0.017 (compare 0.061)
 +
 
 +
control: 0.101 (compare 0.054)
 +
 
 +
The pVJ4 cultures with IPTG and the control did not lyse (the OD’s went up slightly at room temperature as expected)
 +
The pVJ4 culture with arabinose did lyse (the OD significantly decreased overnight)
 +
 
 +
 
 +
Prepared DNA (PCR products) to load in gel
 +
*5ul PCR product
 +
*4ul H20
 +
*1ul loading dye (10x bluejuice)
 +
Can also load 9ul DNA and 1ul bluejuice depending on desired intensity of band
 +
Add 10ul to each well
 +
 
 +
Viewed gel (took picture on gel capture) and saw NO bands
 +
Possible that not enough DNA was run in gel, so re-ran gel with 9ul DNA and 1ul bluejuice
 +
Still so no bands when gel was viewed
 +
 
 +
Will re-run PCR on sunday with the primers and template primers as a positive control
 +
Elongate for 2 minutes
 +
 
 +
Our fragment that we want to amplify is 1320bp without primers.  While the 2nd PCR is running, we will digest a microgram of our DNA at sites that are specified in the published sequence of our cassette:
 +
EcoRI is found at the beginning
 +
EcoRV is found near the middle
 +
Therefore, the digested fragment should be approximately 660bp. We will run the digest product alongside our PCR product to ensure that we have the DNA we think we have.
 +
 
 +
Professor Palmore suggested that we use electrodes made of an inert metal i.e. gold or platinum to reduce the possibility of redox chemistry occurring in our solution.  We spoke to Mr. Ed Mullen in the machine shop who allowed us to have some platinum wire salvaged from electrophoresis boxes
 +
We replaced the copper wire from our old apparatus with platinum wire.

Latest revision as of 00:42, 29 October 2008


Toxipop lab notebook.png

3 July 2008

  • Arabinose was added to mid log phase cultures of pVJ4 (containing the Cell Lysis Cassette and an Arabinose inducible promoter) and pDKL02 (Cell Lysis Cassette and IPTG inducible promoter) and left overnight.
  • Results:
  1. pVJ4 + Arabniose - completely clear solution
  2. pVJ4 + IPTG - lots of growth
  3. pVJ4 alone - lots of growth
  4. pDKL02 + Arabinose - growth
  5. pDKL02 + IPTG - growth
  • The clear solution of pVJ4 and Arabinose was placed in more LB to see if growth would occur. There was no further growth signifying that cell lysis had occurred. The R gene of the cassette degrades the membrane when expressed hence the clearing of the solution.
  • The IPTG solution did not contain lactose, which is probably the reason it did not work


OD measurements after pVJ4 cultures were left over night +IPTG: 0.084 (compare 0.070 last night) +arabinose: 0.017 (compare 0.061)

control: 0.101 (compare 0.054)

The pVJ4 cultures with IPTG and the control did not lyse (the OD’s went up slightly at room temperature as expected) The pVJ4 culture with arabinose did lyse (the OD significantly decreased overnight)


Prepared DNA (PCR products) to load in gel

  • 5ul PCR product
  • 4ul H20
  • 1ul loading dye (10x bluejuice)

Can also load 9ul DNA and 1ul bluejuice depending on desired intensity of band Add 10ul to each well

Viewed gel (took picture on gel capture) and saw NO bands Possible that not enough DNA was run in gel, so re-ran gel with 9ul DNA and 1ul bluejuice Still so no bands when gel was viewed

Will re-run PCR on sunday with the primers and template primers as a positive control Elongate for 2 minutes

Our fragment that we want to amplify is 1320bp without primers. While the 2nd PCR is running, we will digest a microgram of our DNA at sites that are specified in the published sequence of our cassette: EcoRI is found at the beginning EcoRV is found near the middle Therefore, the digested fragment should be approximately 660bp. We will run the digest product alongside our PCR product to ensure that we have the DNA we think we have.

Professor Palmore suggested that we use electrodes made of an inert metal i.e. gold or platinum to reduce the possibility of redox chemistry occurring in our solution. We spoke to Mr. Ed Mullen in the machine shop who allowed us to have some platinum wire salvaged from electrophoresis boxes We replaced the copper wire from our old apparatus with platinum wire.

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