Difference between revisions of "Part:BBa K496002"

 
 
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<partinfo>BBa_K496002 short</partinfo>
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<partinfo>BBa_K496000 short</partinfo>
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[[Image:HokkaidoU_Japan_PCR_Protocol_Fig1.png|right|400px|thumb|Fig. 1]]
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[[Image:HokkaidoU_Japan_PCR_Protocol_Fig2.png|right|150px|thumb|Fig. 2]]
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&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid, or BioBrick is toxic to host cell and can't be amplified. In moments like these, PCR is handy. PCR is also great because it doesn't have many steps, the more steps the more ways to fail.
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&nbsp;&nbsp;&nbsp;When we amplify BioBrick parts or vectors, primers which anneal to prefix and suffix are used, because they are universal. But unlike miniprep products, PCR products don't produce two distinct bands when they are digested. So we are left wondering if digestion went well.
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&nbsp;&nbsp;&nbsp;The iGEM HokkaidoU 2010 created a set of primers which solves this problem. The idea is quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the Biobrick (Fig. 1).
  
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Used for amplifying BioBricks. When plasmid with BioBrick is amplified generates 100bp BioBrick prefix tail (prefix included), and 200bp BioBrick suffix tail (suffix included). So when restriction enzyme digestion is performed correctly this should leave traceable band when electrophoresis is performed. Also DNA cut offs are of different sizes to visualize which end is digested.  
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&nbsp;&nbsp;&nbsp;PCR products that are amplified by using this primer set produces distinct approx 100 bp and 200 bp fragments (Fig. 2).  
  
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This primer set makes amplified BioBricks 300bp longer making small parts like RBS easier to extract from gel. And even when small BioBrick is single enzyme digested it is still longer than 100bp or 200bp accordingly. This primer set is currently experimental.  
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&nbsp;&nbsp;&nbsp;You can judge if digestion was successful by presence of these bands. Compared to the intact PCR product, you will see the significant change in digested PCR fragment length. The fact that all 10 current high copy number assembly vectors containing pSB1C3 share the same annealing sites in the BioBrick flanking region makes this possible.  
  
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Should work with pSB1A3 pSB1A7 pSB1AC3 pSB1AK3 pSB1AT3 pSB1C3 pSB1K3 pSB1T3
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&nbsp;&nbsp;&nbsp;This primer set makes amplified BioBricks 300bp longer. So, this makes small parts like RBS easier to handle. And even when small BioBrick is single enzyme digested it is still longer than 100bp or 200bp accordingly. This primer set is currently experimental.
  
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Does not work with, because one of the primers site is missing. pSB2K3
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K496002 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K496000 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K496002 parameters</partinfo>
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<partinfo>BBa_K496000 parameters</partinfo>
 
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Latest revision as of 15:16, 1 October 2011

Forward primer for visualizing restriction enzyme digestion.

Fig. 1
Fig. 2

   Sometimes standard protocol fails to produce plasmid, or BioBrick is toxic to host cell and can't be amplified. In moments like these, PCR is handy. PCR is also great because it doesn't have many steps, the more steps the more ways to fail.

   When we amplify BioBrick parts or vectors, primers which anneal to prefix and suffix are used, because they are universal. But unlike miniprep products, PCR products don't produce two distinct bands when they are digested. So we are left wondering if digestion went well.

   The iGEM HokkaidoU 2010 created a set of primers which solves this problem. The idea is quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the Biobrick (Fig. 1).

   PCR products that are amplified by using this primer set produces distinct approx 100 bp and 200 bp fragments (Fig. 2).

   You can judge if digestion was successful by presence of these bands. Compared to the intact PCR product, you will see the significant change in digested PCR fragment length. The fact that all 10 current high copy number assembly vectors containing pSB1C3 share the same annealing sites in the BioBrick flanking region makes this possible.

   This primer set makes amplified BioBricks 300bp longer. So, this makes small parts like RBS easier to handle. And even when small BioBrick is single enzyme digested it is still longer than 100bp or 200bp accordingly. This primer set is currently experimental.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]