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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Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't by 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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When amplifying BioBricks primers which anneal to prefix and suffix are used. Good thing about them is that they are universal so with only 2 you can amplify all BioBricks. But unlike miniprep product BioBrick amplified like this don't produce 2 distinct bands when digested. So you are left wondering if digestion went well.  
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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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&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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We created a set of primers which solves this problem. You can judge if digestion was successful by presence of approximatively 100 bp or/and 200 bp bands and significant change in PCRed fragments length.
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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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So idea behind this primers quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the part like in Fig.1 bellow.
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[[Image:HokkaidoU_Japan_PCR_Protocol_Fig1.png|Fig.1]]
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[[Image:HokkaidoU_Japan_PCR_Protocol_Fig2.png|thumb|Fig.2]]
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Now when digested PCRed fragment produces distinct approx 100 bp or/and 200 bp fragment depending on cut sites. Knowing this you can continue to the next step with the sure and light heart (see Fig.2). This is crucial when you take into account that many participants have limited at the bench experience before hand.
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As everyone have guest by now these primers anneal to plasmid regions flanking the BioBrick. And there are more than 40 different primers. But not all of them are quite so different in fact all 7 registries assembly plasmids share the same sequence on the BioBrick flanking regions. And few others do also, bringing the number up to about 10.
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And with the new registry requirement to submit all BioBricks in pSB1C3 plasmid present the possibility that in near future many if not all plasmids could be supported by this primers making assembly easier.
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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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Should work with pSB1A3 pSB1A7 pSB1AC3 pSB1AK3 pSB1AT3 pSB1C3 pSB1K3 pSB1T3
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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]