Labs 5 and 6 Manual

Labs 5 and 6 Manual - WEEK 5: September 27 October 3...

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WEEK 5: September 27 – October 3 Bacterial Transformation, Effect of Washing on Hand Flora Pure Culture Isolation     Check  your  streak  plate  to see  if isolated  colonies  were  obtained  and  if only  one  type  of   colony  appears  on  each  plate.  Again,  if not, how  could  your  technique  be  improved?   * * * Titration of Bacteriophage Results Count  the  plaque s  formed  by  the  phage  by  making  a  mark  with  a  marking  pencil  on  the   bottom  of  the  plate  over  each  plaque  and  counting  as  you  mark.   Count  only  one  set   of  replicate  plates.  You  should  calculate  the  plaque  forming  units  per  milliliter  (PFU/ml)  of the   original  sample  based  on  the  average  number  of  plaque s  on  the  two  plates.  Multiply  the   direct  count  by  the  dilution  used  to obtain  the  PFU per  ml  of stock  culture.   Stock  concentration  (PFU/ml)  = # plaque/volume  plated  (ml)  x 1/concentration  plated For example,  if you  counted  46  plaque s  on  one  of your  10 -3  plates and 40 on the other, you   would average the two, yielding 43 x 10 3  or 4.3 x 10 4  PFU/ml of original stock. Report your calculation  to your TA who will determine an average for the class and communicate that number back to the students.  There is also a demonstration of virus-infected and non-infected cell cultures. The virus kills the cells leaving gaps  in the tissue culture monolayer, with infected cells curling up, demonstrating what is called cytopathic effect  (CPE). Note the difference between the 'lawn' of infected cells versus the uninfected. This CPE is analogous to the  holes in the bacterial cell lawn caused by the phage, except that since the virus is released from a lysed host cell  into the liquid media, the released viruses may infect cells anywhere in the monolayer, not just the adjacent cells as  in your phage plates. * * * MCRO 251 Lab Manual  Exercises to Complete    New Exercises   30
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Bacterial Transformation Introduction Bacterial  transformation  is a  form  of horizontal  gene  transfer  accomplished  through  the  uptake   of ‘naked’  DNA by  certain  bacteria.  Following  uptake,  this  exogenous  DNA is either  maintained   as  an  episomal  plasmid  (such  as  the  plasmid  in today’s  lab)  or  it may  be  incorporated  into  a   homologous   site   in   the   host   bacterium’s   chromoso m e.   Either   results   in   a   recombinant   organism.  Some  species  of bacteria  are  naturally  able  to take  up  and  incorporate  such  foreign   DNA via  transformation  at some  point  in their  cell  cycle  and  are  referred  to  as  ‘competent’.  
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This note was uploaded on 02/11/2012 for the course MCRO 251 taught by Professor Lorrainecramer during the Fall '09 term at UNC.

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Labs 5 and 6 Manual - WEEK 5: September 27 October 3...

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