Difference between revisions of "Part:pSB3C5:Experience"
 
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===Applications of pSB3C5===
 
===Applications of pSB3C5===
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===User Reviews===
 
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<I>Reshma Shetty</I>
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<I>[[User:Sl.ilya|Ilya Vainberg Slutskin]]</I>
 
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This plasmid seems to work.  When transformed into TOP10 cells, pSB3C5-I52001 does not transform but when cut with NotI and self-ligated (to eliminate ccdB), lots of colonies are obtained.  However, when miniprepped, pSB3C5-I52001 does not give as high of yields as expected for a high copy plasmid likely due to mutations in BBa_I52001 that render it nonfunctional as a high copy origin.  I am working to address this issue.   
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[[Part:pSB3C5|pSB3C5]], obtained from 2012 distribution kit well 3C, has consistently gave higher miniprep concentrations in comparison to pSB1C3. Even after taking into account the difference in plasmid size (in bp) the calculated copy number is higher for pSB3C5 than for pSB1C3. This suggests that this plasmid might not function as a medium-low copy plasmid. Similar observations have been found by [[User:Rshetty|Reshma Shetty]] for [[Part:pSB3K5|pSB3K5]], which uses the same replication origin.
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<I>[[User:Rshetty|Reshma Shetty]]</I>
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[[Part:pSB3C5|pSB3C5]] is a functional low to medium copy plasmid but does not propagate at high copy.  When transformed into TOP10 cells, [[Part:pSB3C5|pSB3C5]]-[[Part:BBa_I52001|I52001]] does not transform as expected.  When [[Part:pSB3C5|pSB3C5]]-[[Part:BBa_I52001|I52001]] is cut with NotI,  self-ligated (to eliminate ''ccdB'' positive selection marker) and tranformed into TOP10, lots of colonies are obtained.  However, [[Part:pSB3C5|pSB3C5]]-[[Part:BBa_I52001|I52001]] does not give high miniprep yields due to mutations in [[Part:BBa_I52001|BBa_I52001]] that render it nonfunctional as a high copy origin.  [[Part:pSB3C5|pSB3C5]]-[[Part:BBa_I52001|I52001]] works fine as a plasmid but is not as convenient for plasmid DNA purification.
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<partinfo>BBa_R0051 AddReview 0</partinfo>
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<I> Aberdeen_Scotland 2009 </I>
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The transformation was twice unsuccessful.  Plasmid rescued contained BBa_J04450.
 
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Latest revision as of 09:57, 24 October 2016

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of pSB3C5

User Reviews

UNIQ3ee692e7f9b3f8ec-partinfo-00000000-QINU

Ilya Vainberg Slutskin

pSB3C5, obtained from 2012 distribution kit well 3C, has consistently gave higher miniprep concentrations in comparison to pSB1C3. Even after taking into account the difference in plasmid size (in bp) the calculated copy number is higher for pSB3C5 than for pSB1C3. This suggests that this plasmid might not function as a medium-low copy plasmid. Similar observations have been found by Reshma Shetty for pSB3K5, which uses the same replication origin.

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••••

Reshma Shetty

pSB3C5 is a functional low to medium copy plasmid but does not propagate at high copy. When transformed into TOP10 cells, pSB3C5-I52001 does not transform as expected. When pSB3C5-I52001 is cut with NotI, self-ligated (to eliminate ccdB positive selection marker) and tranformed into TOP10, lots of colonies are obtained. However, pSB3C5-I52001 does not give high miniprep yields due to mutations in BBa_I52001 that render it nonfunctional as a high copy origin. pSB3C5-I52001 works fine as a plasmid but is not as convenient for plasmid DNA purification.

Aberdeen_Scotland 2009

The transformation was twice unsuccessful. Plasmid rescued contained BBa_J04450.

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