Difference between revisions of "Libraries, Vectors and Screening"

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=== An index of general molecular biology techniques used in studies of ''S. purpuratus'' ===
 
=== An index of general molecular biology techniques used in studies of ''S. purpuratus'' ===
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[[array|Arrayed Filter Manual]]<br />   
 
[[array|Arrayed Filter Manual]]<br />   
 
The techniques used to hybridize probes to arrays of cDNAs on filters are detailed here. The manual is based on pages from the RZPD with modifications installed at Caltech.
 
The techniques used to hybridize probes to arrays of cDNAs on filters are detailed here. The manual is based on pages from the RZPD with modifications installed at Caltech.
 +
  
 
[[download/DIGscreen.pdf|Non-radioactive library screening protocol]]<br />
 
[[download/DIGscreen.pdf|Non-radioactive library screening protocol]]<br />
 
The availability of sensitive chemiluminescent detection systems for hybridization screening of arrayed library filters now makes it possible to reach outcomes similar to methods using 32P labeled probes. We have used a digoxygenin based protocol with good success. It is detailed [[download/DIGscreen.pdf|here]].
 
The availability of sensitive chemiluminescent detection systems for hybridization screening of arrayed library filters now makes it possible to reach outcomes similar to methods using 32P labeled probes. We have used a digoxygenin based protocol with good success. It is detailed [[download/DIGscreen.pdf|here]].
 +
  
 
[[subtrmethods|Subtractive Probe Analysis]]<br />
 
[[subtrmethods|Subtractive Probe Analysis]]<br />
 
Our techniques for complex probes to be used on arrayed filters for gene discovery have been perfected by Jonathan Rast in the Davidson Laboratory. The investigation underlying this work is published: Rast J. P. et al, Developmental Biology 228 (2): 270-286, 2000. Supplementary data is presented here, as well.
 
Our techniques for complex probes to be used on arrayed filters for gene discovery have been perfected by Jonathan Rast in the Davidson Laboratory. The investigation underlying this work is published: Rast J. P. et al, Developmental Biology 228 (2): 270-286, 2000. Supplementary data is presented here, as well.
 +
  
 
[[libprepd|Additional Notes on cDNA Library Preparations]]<br />
 
[[libprepd|Additional Notes on cDNA Library Preparations]]<br />
 
The procedures used to construct the cDNA libraries are described here.
 
The procedures used to construct the cDNA libraries are described here.
 +
  
 
[[epgfp|The Reporter Plasmid, EpGFPII]]<br />
 
[[epgfp|The Reporter Plasmid, EpGFPII]]<br />
 
The sequence and map of the reporter construct described in: R. A. Cameron, P. Oliveri, J. Wyllie, and E. H. Davidson (2004) cis-Regulatory Activity of Randomly Chosen Genomic Fragments from the Sea Urchin. Gene Expression Patterns 4, 205-213 can be found here.
 
The sequence and map of the reporter construct described in: R. A. Cameron, P. Oliveri, J. Wyllie, and E. H. Davidson (2004) cis-Regulatory Activity of Randomly Chosen Genomic Fragments from the Sea Urchin. Gene Expression Patterns 4, 205-213 can be found here.
 +
  
 
[[bac_vector_library|BAC Library Vector]]<br />
 
[[bac_vector_library|BAC Library Vector]]<br />
 
The BAC vector pBACe3.6 is the one we have used in the preparation of all of our macro-array genomic libraries. Constructed in Pieter J. de Jong's laboratory at the Children's Hospital Oakland Research Center, it has been used extensively for genomic libraries and sequencing projects of many species.
 
The BAC vector pBACe3.6 is the one we have used in the preparation of all of our macro-array genomic libraries. Constructed in Pieter J. de Jong's laboratory at the Children's Hospital Oakland Research Center, it has been used extensively for genomic libraries and sequencing projects of many species.
  
<span class="newwin">[http://new.echinobase.org/literature/article.do?method=display&articleId=42133 Sea Urchin Fertilization Laboratory]</span><br />
 
Vic Vacquier developed a handout for university students on how to observe sea urchin fertilization in a university lab class setting. All that is needed are sea urchin gametes, a microscope, a few solutions and this handout. The handout works well for both 1 or 2 three hour labs. The student reads through the handout, makes the simple observations, and does the simple, direct experiments to gain insight into the fascinating process of sperm-egg interaction and egg activation.
 
  
 
<span class="newwin">Fluorescent Protein Vectors</span><br />
 
<span class="newwin">Fluorescent Protein Vectors</span><br />
 
The expression of exogenous proteins in sea urchins embryos, by injection of recombinant mRNAs, is routine procedure. The Hamdoun lab has generated a vector set with the major color and tag position variants that is useful for fluorescent protein tagging and expression in urchins. The complete vector set and can be obtained here: [https://www.addgene.org/kits/hamdoun-fluorescent-proteins/]. A description of the vectors can be found in Gokirmak et al., 2012 [http://new.echinobase.org/literature/article.do?method=display&articleId=42631 Vectors for generation of exogenous mRNAs fused to fluorescent proteins for expression in urchins]
 
The expression of exogenous proteins in sea urchins embryos, by injection of recombinant mRNAs, is routine procedure. The Hamdoun lab has generated a vector set with the major color and tag position variants that is useful for fluorescent protein tagging and expression in urchins. The complete vector set and can be obtained here: [https://www.addgene.org/kits/hamdoun-fluorescent-proteins/]. A description of the vectors can be found in Gokirmak et al., 2012 [http://new.echinobase.org/literature/article.do?method=display&articleId=42631 Vectors for generation of exogenous mRNAs fused to fluorescent proteins for expression in urchins]

Latest revision as of 15:37, 4 February 2021


Below is a list of links to common methods. If you would like to add to the list please contact us.


An index of general molecular biology techniques used in studies of S. purpuratus

Arrayed Filter Manual
The techniques used to hybridize probes to arrays of cDNAs on filters are detailed here. The manual is based on pages from the RZPD with modifications installed at Caltech.


Non-radioactive library screening protocol
The availability of sensitive chemiluminescent detection systems for hybridization screening of arrayed library filters now makes it possible to reach outcomes similar to methods using 32P labeled probes. We have used a digoxygenin based protocol with good success. It is detailed here.


Subtractive Probe Analysis
Our techniques for complex probes to be used on arrayed filters for gene discovery have been perfected by Jonathan Rast in the Davidson Laboratory. The investigation underlying this work is published: Rast J. P. et al, Developmental Biology 228 (2): 270-286, 2000. Supplementary data is presented here, as well.


Additional Notes on cDNA Library Preparations
The procedures used to construct the cDNA libraries are described here.


The Reporter Plasmid, EpGFPII
The sequence and map of the reporter construct described in: R. A. Cameron, P. Oliveri, J. Wyllie, and E. H. Davidson (2004) cis-Regulatory Activity of Randomly Chosen Genomic Fragments from the Sea Urchin. Gene Expression Patterns 4, 205-213 can be found here.


BAC Library Vector
The BAC vector pBACe3.6 is the one we have used in the preparation of all of our macro-array genomic libraries. Constructed in Pieter J. de Jong's laboratory at the Children's Hospital Oakland Research Center, it has been used extensively for genomic libraries and sequencing projects of many species.


Fluorescent Protein Vectors

The expression of exogenous proteins in sea urchins embryos, by injection of recombinant mRNAs, is routine procedure. The Hamdoun lab has generated a vector set with the major color and tag position variants that is useful for fluorescent protein tagging and expression in urchins. The complete vector set and can be obtained here: [1]. A description of the vectors can be found in Gokirmak et al., 2012 Vectors for generation of exogenous mRNAs fused to fluorescent proteins for expression in urchins