840:153g:Projects/project23/2012/10/25

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(Continuation of the Mutagenesis Reaction)
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(Continuation of the Mutagenesis Reaction)
 
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==Continuation of the Mutagenesis Reaction ==
==Continuation of the Mutagenesis Reaction ==
-
* On Tuesday, we ran the gel for last week's PCR products.   This was done on a dual-lane chamber, loaded as follows:
+
*On Tuesday, we prepared to combine our upstream and downstream fragments in the final mutagenesis step.  First a high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. We then prepared our samples to amplify. Our approach was to mix the fragments together with or without primers to try to identify an effective combination to create our entire gene.  For each set we also investigated the possible effect of template concentration and annealing temperature, for a total of 12 different combinations.  Our nomenclature used is three-part, and outlined below.
 +
 +
 +
*1.)Primer configuration
 +
**N = no primers used
 +
**P = outside primers used
 +
*2.)Template DNA concentration
 +
** + = 1:20
 +
** - = 1:200
 +
*3.)Annealing temperature for PCR, in °C
 +
 +
*We used the same PCR protocol as last week. 
 +
 +
 +
*On Thursday, we ran the gel for Tuesday's PCR products. 
 +
 +
*Our positive control was standard template DNA and associated primers from lab stock
 +
*Our negative control had outside primers and no template
 +
 +
 +
*We loaded our samples as follows:
*Top Lane:
*Top Lane:
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-
*Bottom Lane:
+
*Lower Lane:
#100 bp ladder
#100 bp ladder
#N+55
#N+55
#N+60
#N+60
#N+65
#N+65
-
#Positive Control
+
#Negative Control
#N-55
#N-55
#N-60
#N-60
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-
*We ran these on a 1% agarose gel for 30min at 120V
+
*We ran these samples on a 1% agarose gel for 30min at 120V
*Results:
*Results:
 +
 +
 +
*Upper Lane
 +
[[Image:10.25.12_Primered_Lanes.jpg|600px]]
 +
 +
 +
*Lower Lane
 +
[[Image:10.25.12_Non-primered_Lanes.jpg|600px]]
 +
 +
 +
*Conclusions:
 +
**Our positive control checked ok, indicating working PCR reagents
 +
**Our negative control showed a faint band in the <100bp region, likely due to primers.  Nothing else appeared, indicating an uncontaminated working stock.
 +
**Our non-primered set did not work.  This should, in theory, so perhaps we need to adjust the thermocycle protocol.
 +
**Our primered set did amplify a numer of distinct bands. Our target length of ~400bp showed bands on both the upper and lower boundaries.  One of these may be our intended product, as there is some range of error in marker ladders.  Perhaps none of the bands are our product, however, and are just PCR artifacts.
 +
**Template dilution works well for either high or low concentrations.  We will use 1:200 for further amplification.
 +
**Annealing temperature did not seem to vary results for this range.
 +
 +
 +
*After analyzing this gel, we decided to run another PCR to do a side-by-side comparison of the two fragments and the combined reaction, to see if they are separate products.  We used only the 1:200 template, and the 65°C annealing temperature, as they gave us the cleanest images to date.  We prepared an upstream and downstream reaction from our original total-DNA sample, and primered/ non-primered set identical to earlier this week.  We also prepared each of these sets without template DNA for negative control.  Lastly, we prepared 6 more primered samples to extract DNA from if we prove to have our intended product. We cycled our PCR with the same protocol we used in the past, but with a non-gradient annealing temperature.
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__NOTOC__
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[[category:OWWLabNotebookV1]]
[[category:OWWLabNotebookV1]]

Current revision

Cloning of the OmcF gene Main project page
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Continuation of the Mutagenesis Reaction

  • On Tuesday, we prepared to combine our upstream and downstream fragments in the final mutagenesis step. First a high concentration of 1:20 template DNA, and a low concentration of 1:200 were prepared with water for each the upstream and downstream fragments. We then prepared our samples to amplify. Our approach was to mix the fragments together with or without primers to try to identify an effective combination to create our entire gene. For each set we also investigated the possible effect of template concentration and annealing temperature, for a total of 12 different combinations. Our nomenclature used is three-part, and outlined below.


  • 1.)Primer configuration
    • N = no primers used
    • P = outside primers used
  • 2.)Template DNA concentration
    • + = 1:20
    • - = 1:200
  • 3.)Annealing temperature for PCR, in °C
  • We used the same PCR protocol as last week.


  • On Thursday, we ran the gel for Tuesday's PCR products.
  • Our positive control was standard template DNA and associated primers from lab stock
  • Our negative control had outside primers and no template


  • We loaded our samples as follows:
  • Top Lane:
  1. 100 bp ladder
  2. P+55
  3. P+60
  4. P+65
  5. Positive Control
  6. P-55
  7. P-60
  8. P-65
  9. 100 bp ladder


  • Lower Lane:
  1. 100 bp ladder
  2. N+55
  3. N+60
  4. N+65
  5. Negative Control
  6. N-55
  7. N-60
  8. N-65
  9. 100 bp ladder


  • We ran these samples on a 1% agarose gel for 30min at 120V


  • Results:


  • Upper Lane


  • Lower Lane


  • Conclusions:
    • Our positive control checked ok, indicating working PCR reagents
    • Our negative control showed a faint band in the <100bp region, likely due to primers. Nothing else appeared, indicating an uncontaminated working stock.
    • Our non-primered set did not work. This should, in theory, so perhaps we need to adjust the thermocycle protocol.
    • Our primered set did amplify a numer of distinct bands. Our target length of ~400bp showed bands on both the upper and lower boundaries. One of these may be our intended product, as there is some range of error in marker ladders. Perhaps none of the bands are our product, however, and are just PCR artifacts.
    • Template dilution works well for either high or low concentrations. We will use 1:200 for further amplification.
    • Annealing temperature did not seem to vary results for this range.


  • After analyzing this gel, we decided to run another PCR to do a side-by-side comparison of the two fragments and the combined reaction, to see if they are separate products. We used only the 1:200 template, and the 65°C annealing temperature, as they gave us the cleanest images to date. We prepared an upstream and downstream reaction from our original total-DNA sample, and primered/ non-primered set identical to earlier this week. We also prepared each of these sets without template DNA for negative control. Lastly, we prepared 6 more primered samples to extract DNA from if we prove to have our intended product. We cycled our PCR with the same protocol we used in the past, but with a non-gradient annealing temperature.
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