Team:Rice University/Notebook/20 June 2008

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(Difference between revisions)
(New page: =Friday, 20 June= *Taylor Stevenson: **VCS257 SupA+ cells were grown O/N @ 37*C in Lambda media. 4.5mL culture was infected with 4.5uL of phage packaging product from November, 2007 and a...)
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=Friday, 20 June=
=Friday, 20 June=
*Taylor Stevenson:
*Taylor Stevenson:
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**VCS257 SupA+ cells were grown O/N @ 37*C in Lambda media.  4.5mL culture was infected with 4.5uL of phage packaging product from November, 2007 and allowed to incubate for 20min @ 37*C shaking @ 250 rpm.  Infected culture was heat shocked @ 42*C for 20min, inducing lysis by denaturing the CI857 repressor.  Cells were then grown @ 37*C shaking @ 250 rpm, and their optical density was measured for XXXXX hours.
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**Lysis Assay of VCS257 SupA+  
 +
*#Cells were grown O/N @ 37*C in Lambda media.   
 +
*#4.5mL culture was infected with 4.5uL of phage packaging product from November, 2007 and allowed to incubate for 20min @ 37*C shaking @ 250 rpm.  Infected culture was heat shocked @ 42*C for 20min, inducing lysis by denaturing the CI857 repressor.   
 +
*#Cells were then grown and their optical density was measured at different time points after heat shock.
 +
**Lysogeny Screen - Results
 +
*#An infected VCS257 culture was streaked onto LB-agar.  Isolated colonies were used to innoculate a 96 deep-well plate which was grown O/N @ 30*C shaking @ 300rpm.  Using the well replicator, each of the wells was spotted onto two large petri dishes.  One dish was grown @ 30*C and the other was grown @ 42*C O/N. Spots that grew on 30*C plates and not on 42*C plates are lysogens as the phage infected cells would lyse due to the temperature sensitive cI.  The plates show that there is only one colony that could be a possible lysogen.
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*# The possible lysogen colony was streaked onto LB plates to be grown O/N @ 30*C and 42*C to verify temperature screen.
 +
*# A colony PCR was performed on the possible lysogen using the CroF and CroR primers.  If a copy of the lambda genome is incorporated into the lysogen's, the PCR should result in amplification of the phage gene ''cro'' and produce a 400bp product.  Additionally, three other PCR reactions were performed in parallel.
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*## -negative control using water
 +
*## -positive control using lambda DNA
 +
*## -second sample using lambda particles from stocks

Revision as of 18:04, 20 June 2008

Friday, 20 June

  • Taylor Stevenson:
    • Lysis Assay of VCS257 SupA+
    1. Cells were grown O/N @ 37*C in Lambda media.
    2. 4.5mL culture was infected with 4.5uL of phage packaging product from November, 2007 and allowed to incubate for 20min @ 37*C shaking @ 250 rpm. Infected culture was heat shocked @ 42*C for 20min, inducing lysis by denaturing the CI857 repressor.
    3. Cells were then grown and their optical density was measured at different time points after heat shock.
    • Lysogeny Screen - Results
    1. An infected VCS257 culture was streaked onto LB-agar. Isolated colonies were used to innoculate a 96 deep-well plate which was grown O/N @ 30*C shaking @ 300rpm. Using the well replicator, each of the wells was spotted onto two large petri dishes. One dish was grown @ 30*C and the other was grown @ 42*C O/N. Spots that grew on 30*C plates and not on 42*C plates are lysogens as the phage infected cells would lyse due to the temperature sensitive cI. The plates show that there is only one colony that could be a possible lysogen.
    2. The possible lysogen colony was streaked onto LB plates to be grown O/N @ 30*C and 42*C to verify temperature screen.
    3. A colony PCR was performed on the possible lysogen using the CroF and CroR primers. If a copy of the lambda genome is incorporated into the lysogen's, the PCR should result in amplification of the phage gene cro and produce a 400bp product. Additionally, three other PCR reactions were performed in parallel.
      1. -negative control using water
      2. -positive control using lambda DNA
      3. -second sample using lambda particles from stocks
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