Echinobase/bac information: Difference between revisions
imported>Ctelmer |
No edit summary |
||
| (6 intermediate revisions by 2 users not shown) | |||
| Line 2: | Line 2: | ||
== Genomic BAC Resources == | == Genomic BAC Resources == | ||
Libraries of genomic sequence fragments are maintained in bacterial artificial chromosome (BAC) vectors for seven lower deuterostome species ([https://wiki.echinobase.org/echinowiki/index.php/BAC_Table BAC Table]). These libraries were originally constructed as part of the Sea Urchin Genome Project. On average, the insert sizes for our BAC libraries are ~140 kb. Thus, for the 800 Mb ''S. purpuratus'' genome, 1X coverage is encompassed by, on average, 5,700 clones. Each library consists of >100,000 clones, providing ~17X genome coverage. | |||
A subset of the ''S. purpuratus'' BACs have been mapped to the genome assembly. As part of the SUGP, about 8,000 BAC clones were end sequenced using Sanger technology. Genomic positions for these BACs are indicated as a track in JBrowse. An additional set of individual BACs were sequenced completely; links to the NCBI page are provided in the [https://wiki.echinobase.org/echinowiki/index.php/BAC_Table BAC Table]. Finally, some BACs have been identified using hybridization screening of arrayed library filters (see | A subset of the ''S. purpuratus'' BACs have been mapped to the v3.1 genome assembly. As part of the SUGP, about 8,000 BAC clones were end sequenced using Sanger technology. Genomic positions for these BACs are indicated as a track in JBrowse on the v5.0 genome assembly. An additional set of individual BACs were sequenced completely; links to the NCBI page are provided in the [https://wiki.echinobase.org/echinowiki/index.php/BAC_Table BAC Table]. Finally, some BACs have been identified using hybridization screening of arrayed library filters (see [https://wiki.echinobase.org/echinowiki/index.php/BAC_Table BAC Table]). | ||
'''BAC Vector Sequence.''' The BACs are maintained in the pBACe3.6 vector which originates from Children's Hospital Oakland Research Institute in Oakland, California, USA. This vector confers resistance to the antibiotic chloramphenicol and is fully described by Frengen and | <br>'''BAC Vector Sequence.''' The BACs are maintained in the pBACe3.6 vector which originates from Children's Hospital Oakland Research Institute in Oakland, California, USA. This vector confers resistance to the antibiotic chloramphenicol and is fully described by Frengen and colleagues. The vector sequence is available [https://wiki.echinobase.org/echinowiki/index.php/Bac_vector_library here]. | ||
'''Relevant publications:''' | <br>'''Relevant publications:''' | ||
A sea urchin genome project: Sequence scan, virtual map, and additional resources. [http://www.pnas.org/content/97/17/9514.full Cameron, RA, et al., 2000. Proc. Natl. Acad. Sci. USA.] | A sea urchin genome project: Sequence scan, virtual map, and additional resources. [http://www.pnas.org/content/97/17/9514.full Cameron, RA, et al., 2000. Proc. Natl. Acad. Sci. USA.] | ||
Bacterial artificial chromosomes as recombinant reporter constructs to investigate gene expression and regulation in echinoderms. [http:// | Bacterial artificial chromosomes as recombinant reporter constructs to investigate gene expression and regulation in echinoderms. [http://https://www.echinobase.org/literature/article.do?method=display&articleId=45819 Buckley, KM et al., 2017. Briefings in Functional Genomics]. | ||
[https://wiki.echinobase.org/echinowiki/index.php/Bac_vector_library'''BAC Protocols'''] | <br><p>[https://wiki.echinobase.org/echinowiki/index.php/Bac_vector_library'''BAC Protocols''']</p> | ||
Screening a BAC library | <p>Screening a BAC library</p> | ||
Generating a recombinant BAC | <p>Generating a recombinant BAC</p> | ||
BAC miniprep protocol | <p>BAC miniprep protocol</p> | ||
Analyzing BAC DNA by PFGE | <p>Analyzing BAC DNA by PFGE</p> | ||
'''Recombinant BAC Resources''' | <br>'''Recombinant BAC Resources''' | ||
Echinobase has generated and maintains a set of reporter BAC constructs in which a portion of the gene of interest is replaced by the sequence encoding a fluorescent protein (i.e. GFP, mCherry | Echinobase has generated and maintains a set of reporter BAC constructs in which a portion of the gene of interest is replaced by the sequence encoding a fluorescent protein (i.e. GFP, mCherry). Existing clones are available by request. We will also consider requests to make additional recombinant reporter constructs as time permits. | ||
To request a recombinant or wildtype BAC for non-commercial use: | <p>To request a recombinant or wildtype BAC for non-commercial use:</p> | ||
1. Identify the BAC of interest computationally or by screening our libraries | <p>1. Identify the BAC of interest computationally or by screening our libraries.</p> | ||
2. Request the BAC by emailing Veronica Hinman. | <p>2. Request the BAC by emailing Veronica Hinman.</p> | ||
<br><p>'''BAC numbers eg. Sp_117A03_L'''</p> | |||
<p>Sp = Species Sp, ''S. purpuratus''</p> | |||
<p>117 = Plate number</p> | |||
<p>A = Plate row</p> | |||
<p>03 = Plate column</p> | |||
<p>L = long or S, short</p> | |||
<br><p>'''Storing BACs'''</p> | |||
'''Storing BACs''' | |||
'''Short term''' | '''Short term''' | ||
<p>To avoid shearing the long BAC DNA fragments, isolated BACs should never be vortexed or frozen. Store BAC DNA at 4°C in TE (10 mM Tris, pH = 7.4; 10 mM EDTA).</p> | |||
'''Antibiotic conditions''' for working with BAC clones | '''Long term storage''' | ||
Kanamycin | <p>Store glycerol bacteria stocks (15-25% glycerol) at -80°C.</p> | ||
Stock (25 μg/μL): Dissolve 0.5 g of kanamycin into 10 ml of ddH2O. Filter through a 0.22 µm filter to sterilize. Aliquot and store at -20°C. | |||
Use at 1:1000 dilution (25 μg/mL). | <br><p>'''Working with BACs'''</p> | ||
Chloramphenicol | |||
Stock (25 μg/μl): Dissolve in ETOH store at - | <p>'''Antibiotic conditions''' for working with BAC clones</p> | ||
Use at 1:2000 dilution | <p>Kanamycin Stock (25 μg/μL): Dissolve 0.5 g of kanamycin into 10 ml of ddH2O.</p> | ||
<p>Filter through a 0.22 µm filter to sterilize.</p> | |||
<p>Aliquot and store at -20°C.</p> | |||
<p>Use at 1:1000 dilution (25 μg/mL).</p> | |||
<p>Chloramphenicol Stock (25 μg/μl): Dissolve in ETOH store at -20°C.</p> | |||
<p>Use at 1:2000 dilution (12.5 μg/ml).</p> | |||
<br>'''Transport to Pittsburgh''' | |||
<p>The BAC libraries were prepared using the Qbot.</p> | |||
<p style="margin-left: 20px">[[File:Qbot.JPG|frameless|200px]]</p> | |||
<p>The libraries fit into six -80°C freezers.</p> | |||
<p style="margin-left: 20px">[[File:Freezer rm.JPG|frameless|200px]]</p> | |||
<p>The truck had power outlets to plug the freezers for the cross-country drive.</p> | |||
<p style="margin-left: 20px">[[File:Freezer_truck.png|frameless|200px]]</p> | |||
<p>The truck was loaded.</p> | |||
<p style="margin-left: 20px">[[File:Freezer_truck_inside.png|frameless|200px]]</p> | |||
<p>Ready to go!</p> | |||
<p style="margin-left: 20px">[[File:Truck_loaded.png|frameless|200px]]</p> | |||
<p>The CIT Team.</p> | |||
<p style="margin-left: 20px">[[File:CIT group_8956mrgsmall.jpg|frameless|200px]]</p> | |||
Latest revision as of 13:53, 11 March 2022
Genomic BAC Resources
Libraries of genomic sequence fragments are maintained in bacterial artificial chromosome (BAC) vectors for seven lower deuterostome species (BAC Table). These libraries were originally constructed as part of the Sea Urchin Genome Project. On average, the insert sizes for our BAC libraries are ~140 kb. Thus, for the 800 Mb S. purpuratus genome, 1X coverage is encompassed by, on average, 5,700 clones. Each library consists of >100,000 clones, providing ~17X genome coverage.
A subset of the S. purpuratus BACs have been mapped to the v3.1 genome assembly. As part of the SUGP, about 8,000 BAC clones were end sequenced using Sanger technology. Genomic positions for these BACs are indicated as a track in JBrowse on the v5.0 genome assembly. An additional set of individual BACs were sequenced completely; links to the NCBI page are provided in the BAC Table. Finally, some BACs have been identified using hybridization screening of arrayed library filters (see BAC Table).
BAC Vector Sequence. The BACs are maintained in the pBACe3.6 vector which originates from Children's Hospital Oakland Research Institute in Oakland, California, USA. This vector confers resistance to the antibiotic chloramphenicol and is fully described by Frengen and colleagues. The vector sequence is available here.
Relevant publications:
A sea urchin genome project: Sequence scan, virtual map, and additional resources. Cameron, RA, et al., 2000. Proc. Natl. Acad. Sci. USA.
Bacterial artificial chromosomes as recombinant reporter constructs to investigate gene expression and regulation in echinoderms. Buckley, KM et al., 2017. Briefings in Functional Genomics.
Screening a BAC library
Generating a recombinant BAC
BAC miniprep protocol
Analyzing BAC DNA by PFGE
Recombinant BAC Resources
Echinobase has generated and maintains a set of reporter BAC constructs in which a portion of the gene of interest is replaced by the sequence encoding a fluorescent protein (i.e. GFP, mCherry). Existing clones are available by request. We will also consider requests to make additional recombinant reporter constructs as time permits.
To request a recombinant or wildtype BAC for non-commercial use:
1. Identify the BAC of interest computationally or by screening our libraries.
2. Request the BAC by emailing Veronica Hinman.
BAC numbers eg. Sp_117A03_L
Sp = Species Sp, S. purpuratus
117 = Plate number
A = Plate row
03 = Plate column
L = long or S, short
Storing BACs
Short term
To avoid shearing the long BAC DNA fragments, isolated BACs should never be vortexed or frozen. Store BAC DNA at 4°C in TE (10 mM Tris, pH = 7.4; 10 mM EDTA).
Long term storage
Store glycerol bacteria stocks (15-25% glycerol) at -80°C.
Working with BACs
Antibiotic conditions for working with BAC clones
Kanamycin Stock (25 μg/μL): Dissolve 0.5 g of kanamycin into 10 ml of ddH2O.
Filter through a 0.22 µm filter to sterilize.
Aliquot and store at -20°C.
Use at 1:1000 dilution (25 μg/mL).
Chloramphenicol Stock (25 μg/μl): Dissolve in ETOH store at -20°C.
Use at 1:2000 dilution (12.5 μg/ml).
Transport to Pittsburgh
The BAC libraries were prepared using the Qbot.
The libraries fit into six -80°C freezers.
The truck had power outlets to plug the freezers for the cross-country drive.
The truck was loaded.
Ready to go!
The CIT Team.
