mirror of
https://github.com/scinote-eln/scinote-web.git
synced 2024-09-21 23:46:21 +08:00
1102 lines
45 KiB
JSON
1102 lines
45 KiB
JSON
{
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"experiment": {
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"id": 422,
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"name": "CRISPR transformations with Agrobacterium tumefaciens",
|
||
"description": "This template will allow you to do CRISPR transformation of plants with Agrobacterium tumefaciens. The clustered regularly interspaced short palindromic repeats(CRISPR) associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from the bacterial immune system. This technique enables precise genomic modifications in many different organisms and tissues.",
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"uuid": "1a3d39cf-ea51-48a8-b622-d7a9492fd1e7"
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{
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"my_module": {
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"id": 2089,
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"name": "Design and construction of gene-specific sgRNA",
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"id": 5991,
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"protocol": {
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"steps": [
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{
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"step": {
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"id": 4261,
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"name": "Use the online tools",
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"text": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Paste selected sequence in the selected online tool. <br><br>Parameters that should be considered are:<br>\n</p>\n<ul>\n<li style=\"text-align: justify;\">target length</li>\n<li style=\"text-align: justify;\">the maximum number of tandemly repeated nucleotides</li>\n<li style=\"text-align: justify;\">minimum/maximum GC content. </li>\n</ul>\nSelect a genome to query for potential off-target recognition and analyze it. Evaluate and prioritize targets using sequence identity as well as off-target sequence identity.</body></html>"
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"text": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Consider choosing <strong>several different target sites</strong>, since not all sgRNAs work equally well. <br>Consider using a <strong>20 bp target site that also contains a recognition site of a commercially available restriction enzyme</strong>. The target site of the restriction enzyme should span the site of Cas9 cleavage (<strong>3 bp</strong> in front of the PAM). This will allow you to use the restriction enzyme length polymorphism assay to detect mutations in your <em>gene of interest.</em></p></body></html>"
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},
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"position": 0
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"step": {
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"name": "Selecting genomic DNA region",
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"asset": {
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"id": 3755,
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"created_at": "2019-02-20T07:40:21.348Z",
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"updated_at": "2019-02-20T07:40:29.583Z",
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},
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}
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"text": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Genomic DNA region must correspond to the crRNA sequence of the sgRNA:<br><br><strong>The 3' end of the DNA target sequence must have a PAM sequence</strong>. The <strong>20 nucleotides</strong> upstream of the PAM sequence will be your targeting sequence (crRNA), and Cas9 nuclease will cleave approximately three bases upstream of the PAM.</p>\n<ol>\n<li>The PAM sequence itself is absolutely required for cleavage, but it is NOT part of the sgRNA sequence and therefore should not be included in the sgRNA.</li>\n<li>The target sequence can be on either DNA strand.</li>\n</ol>\n</body></html>"
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},
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"position": 0
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}
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}
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],
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"results": [
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]
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},
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{
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"my_module": {
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"id": 2090,
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"name": "Subcloning of the construct in plasmid",
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{
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"id": 5992,
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"input_id": 2208,
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}
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"step": {
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"text": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Verify eight <em>E. coli</em> colonies by colony PCR using primer and your protospacer primer with the following setup.<br><br><strong>PCR program:</strong></p>\n<ul>\n<li>95°C 2 min</li>\n<li>(35 cycles) 95°C 30 sec; 51°C 30 sec; 72°C 15 sec </li>\n<li>72°C 15 sec</li>\n<li>72°C 5 min</li>\n<li>20°C </li>\n</ul>\n<br><br>Inoculate positive colony in LB medium supplemented with Amp and grow overnight.</body></html>"
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"text": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Transform competent <em>E. coli </em>cells with the ligation reaction and spread the transformed cells on LB agar plates supplemented with Amp.<br><br><br><strong>Recipe:</strong><br>15 g/L Bacto-tryptone <br>5 g/L yeast extract <br>5 g/L NaCl <br>with 200 μg/mL ampicillin</p></body></html>"
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"text": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Digesting plasmid with a restriction enzyme for <strong>3 hours at 37°C</strong>. Run it on an agarose gel and extract the vector backbone from the gel using the DNA Purification Kit.</p></body></html>"
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},
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{
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||
"step": {
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||
"id": 4574,
|
||
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||
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||
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||
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