User:Karmella Haynes/Notebook/BioBrick cloning/2015/10/08: Difference between revisions
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==10/09/15== | ==10/09/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Cas-tone- H3 inserts into MV12 (MV11-GFP) | * Cas-tone- H3 inserts into MV12 (new MV11-GFP) | ||
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** '''DONE''' PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer) | ** '''DONE''' PCR-amplify histone parts as XbaI-Kozak-histone part-a-NotI. Note: Do no use Kozak BioBrick, since this will add extra amino acids onto conserved histone N-terminal tail. Instead, use the Kozak-like seq. from the Addgene plasmid, +3 extra Kozak bases (included in primer) | ||
* STAGE 3 - Cas-fusion | * STAGE 3 - Cas-fusion | ||
** '''Insert Kozak-histone parts (X/N) into dCas9 plasmid "MV12" (S/N)''' | ** '''Insert Kozak-histone parts (X/N) into dCas9 plasmid + GFP reporter "MV12" (S/N)''' | ||
* STAGE 4 - gRNA | * STAGE 4 - gRNA | ||
** Put pre-existing gRNA (from luc experiment) into pSPgRNA | ** Put pre-existing gRNA (from luc experiment) into pSPgRNA | ||
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'''Assemblies''' | '''Assemblies''' | ||
# H3-1- | # H3-1-40_MV12: H3-1-40/(X/N)/129 + '''MV12/(S/N)/10305''' | ||
# H3-1- | # H3-1-60_MV12: H3-1-60/(X/N)/189 + " | ||
# H3-1- | # H3-1-80_MV12: H3-1-80/(X/N)/250 + " | ||
# H3-1- | # H3-1-116_MV12: H3-1-116/(X/N)/373 + " | ||
# H3-1- | # H3-1-136_MV12: H3-1-136/(X/N)/433 + " | ||
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| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
|- | |- | ||
| 1. MV11 (S/N) || | | 1. MV11 (S/N) || ### || ### || ### | ||
|- | |- | ||
| 2. H3-1-40 PCR || 0.03 || 1.879 || 29.8 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15] | | 2. H3-1-40 PCR || 0.03 || 1.879 || 29.8 [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2015/06/23 06/23/15] | ||
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* Ligations | * Ligations | ||
** > 2:1 ratio calculations... | ** > 2:1 ratio calculations... | ||
** 433 bp largest insert / | ** 433 bp largest insert / 10305 bp vector * 2 * 50 ng vector = 4.2 ng | ||
** 129 bp smallest insert / | ** 129 bp smallest insert / 10305 bp vector * 2 * 50 ng vector = 1.3 ng | ||
{| {{table}} cellspacing="3" <!-- Ligation rxn table --> | {| {{table}} cellspacing="3" <!-- Ligation rxn table --> |
Revision as of 18:08, 8 October 2015
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10/09/15
Cas-tone Project
RESULTS (10/09/15)
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