840:153g:Projects/project27/2012/11/06

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(Transformation and PCR)
Current revision (17:49, 27 November 2012) (view source)
(Transformation and PCR)
 
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* Attempted the transformation of competent ''E. coli'' cells with the ligation products from 10/30 (J61002 plasmid backbone with J23100 insert, and pSB1K3 plasmid backbone with J23100 insert).  After transformation, cells were plated on SB media containing the appropriate antibiotic, along with positive control plated on ampicillin and negative control plated on ampicillin, kanamycin, and no antibiotic.  Untransformed cells were also plated on ampicillin, kanamycin, and no antibiotic.
* Attempted the transformation of competent ''E. coli'' cells with the ligation products from 10/30 (J61002 plasmid backbone with J23100 insert, and pSB1K3 plasmid backbone with J23100 insert).  After transformation, cells were plated on SB media containing the appropriate antibiotic, along with positive control plated on ampicillin and negative control plated on ampicillin, kanamycin, and no antibiotic.  Untransformed cells were also plated on ampicillin, kanamycin, and no antibiotic.
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* PCR results from 11/1 were checked on a gel.  Results were inconclusive, as template DNA amplified with the mmoXY primers and mmoBDZC primers at 55, 60, and 65 deg. showed smearing with expected band sizes being faint or none.   
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* PCR results from 11/1 were checked on a gel.  Results were inconclusive, as lanes showed smearing with expected band sizes being faint or none.  Negative controls showed no results and positive control showed a distinct band at ~500bp, indicating that there was no error in the PCR procedure.
[[Image:!mmoPCR2.jpg]]
[[Image:!mmoPCR2.jpg]]
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* Lanes
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  Lanes
   1-3: mmoBZDC primers (target 2378bp) at 55, 60, 65 degrees, respectively
   1-3: mmoBZDC primers (target 2378bp) at 55, 60, 65 degrees, respectively
     4: negative control (no primers used)
     4: negative control (no primers used)
 +
    5: Marker
 +
  6-8: mmoXY primers (target 2893bp) at 55, 60, 65 degrees, respectively
 +
    9: negative control (no primers used)
 +
    10: positive control (known template and primer set)
* A second PCR was designed in which both the concentration of template DNA and annealing temperature are to be tested.  The PCR protocol will be changed so that three template concentrations will be used for each primer set; a 1:10 dilution of template, 2ul of undiluted template, and 5ul of undiluted template.  These will be annealed at 60 deg. and will be to test the effect of template concentration on amplification.  Also, lower annealing temperatures will be tested by running the original template concentration at 40, 45, and 50 deg. for each primer set.
* A second PCR was designed in which both the concentration of template DNA and annealing temperature are to be tested.  The PCR protocol will be changed so that three template concentrations will be used for each primer set; a 1:10 dilution of template, 2ul of undiluted template, and 5ul of undiluted template.  These will be annealed at 60 deg. and will be to test the effect of template concentration on amplification.  Also, lower annealing temperatures will be tested by running the original template concentration at 40, 45, and 50 deg. for each primer set.

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Transformation and PCR

  • Attempted the transformation of competent E. coli cells with the ligation products from 10/30 (J61002 plasmid backbone with J23100 insert, and pSB1K3 plasmid backbone with J23100 insert). After transformation, cells were plated on SB media containing the appropriate antibiotic, along with positive control plated on ampicillin and negative control plated on ampicillin, kanamycin, and no antibiotic. Untransformed cells were also plated on ampicillin, kanamycin, and no antibiotic.
  • PCR results from 11/1 were checked on a gel. Results were inconclusive, as lanes showed smearing with expected band sizes being faint or none. Negative controls showed no results and positive control showed a distinct band at ~500bp, indicating that there was no error in the PCR procedure.

Image:!mmoPCR2.jpg

  Lanes
  1-3: mmoBZDC primers (target 2378bp) at 55, 60, 65 degrees, respectively
    4: negative control (no primers used)
    5: Marker
  6-8: mmoXY primers (target 2893bp) at 55, 60, 65 degrees, respectively
    9: negative control (no primers used)
   10: positive control (known template and primer set)
  • A second PCR was designed in which both the concentration of template DNA and annealing temperature are to be tested. The PCR protocol will be changed so that three template concentrations will be used for each primer set; a 1:10 dilution of template, 2ul of undiluted template, and 5ul of undiluted template. These will be annealed at 60 deg. and will be to test the effect of template concentration on amplification. Also, lower annealing temperatures will be tested by running the original template concentration at 40, 45, and 50 deg. for each primer set.


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