Difference between revisions of "Part:BBa K784023:Experience"
 
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To test the MCS, we digested 500ng of  plasmid with each of the restriction enzymes. The restriction products were ran  on a gel along with an uncut plasmid. The results are presented in <strong><em>Figure  1</em></strong>.</p>
 
To test the MCS, we digested 500ng of  plasmid with each of the restriction enzymes. The restriction products were ran  on a gel along with an uncut plasmid. The results are presented in <strong><em>Figure  1</em></strong>.</p>
 
<p>It can be noticed that all the enzymes cut efficiently except for XbaI.  After looking again at the sequence of the MCS it was found that the  combination of the overlapping XbaI and BglII sites created a GATC methylation  site. XbaI digestion is affected by this methylation. Therefore, to achieve  highest restriction efficiency with XbaI in this construct the plasmid should  be propagated in <em>dam-</em> <em>E. coli</em> strains. A future improvement to  this MCS would be restoring the original BioBrick prefix by inserting a G downstream  to the XbaI site. This will destroy the BglII site but it will also destroy the  GATC sequence, therefore, restoring the XbaI restriction site to a non  methylated state.</p>
 
<p>It can be noticed that all the enzymes cut efficiently except for XbaI.  After looking again at the sequence of the MCS it was found that the  combination of the overlapping XbaI and BglII sites created a GATC methylation  site. XbaI digestion is affected by this methylation. Therefore, to achieve  highest restriction efficiency with XbaI in this construct the plasmid should  be propagated in <em>dam-</em> <em>E. coli</em> strains. A future improvement to  this MCS would be restoring the original BioBrick prefix by inserting a G downstream  to the XbaI site. This will destroy the BglII site but it will also destroy the  GATC sequence, therefore, restoring the XbaI restriction site to a non  methylated state.</p>
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See [http://2012.igem.org/Team:Technion/Project/Phage#pSB1C3.2BMCS our wiki] for additional info.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 20:29, 26 September 2012

Experience of BBa_K784023

Figure 1: pSB1C3+MCS digested with various unique restriction enzymes. The expected product is 2096bp.

The MCS was constructed by DNA hybridization of two single stranded DNA molecules. The hybridized product was cloned into pSB1C3 using the XbaI and PstI1 sites. The MCS contains the following unique restriction sites: EcoRI, XbaI, BglII, HindIII, PacI, BamHI, SpeI and PstI.
To test the MCS, we digested 500ng of plasmid with each of the restriction enzymes. The restriction products were ran on a gel along with an uncut plasmid. The results are presented in Figure 1.

It can be noticed that all the enzymes cut efficiently except for XbaI. After looking again at the sequence of the MCS it was found that the combination of the overlapping XbaI and BglII sites created a GATC methylation site. XbaI digestion is affected by this methylation. Therefore, to achieve highest restriction efficiency with XbaI in this construct the plasmid should be propagated in dam- E. coli strains. A future improvement to this MCS would be restoring the original BioBrick prefix by inserting a G downstream to the XbaI site. This will destroy the BglII site but it will also destroy the GATC sequence, therefore, restoring the XbaI restriction site to a non methylated state.

See [http://2012.igem.org/Team:Technion/Project/Phage#pSB1C3.2BMCS our wiki] for additional info.

User Reviews

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