User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/23: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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| colspan="2"| | | colspan="2"| | ||
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<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Ryan - Receiver cloning stage 2 | * Ryan - Receiver cloning stage 2 | ||
* | * Rene - sequencing for g053-2, g054 | ||
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* Stage 2 - insert Promoters | * Stage 2 - insert Promoters | ||
** '''Cut & dephos promoters with E/X''' | ** '''Cut & dephos promoters with E/X''' | ||
** Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling | ** '''Insert promoters(E/X) into Vector/Regulator (BbsI) via Lig/Dig cycling''' | ||
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| Sample || OD260 || 260/280 || ng/μL | | Sample || OD260 || 260/280 || ng/μL | ||
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| 1. | | 1. AubR/MRV || 0.258 || 1.906 || 257.7 | ||
|- | |- | ||
| 2. | | 2. BjaR/MRV || 0.177 || 1.904 || 177.3 | ||
|- | |- | ||
| 3. | | 3. RpaR/MRV || 0.246 || 1.896 || 246.2 | ||
|} | |} | ||
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* Digests (Fermentas FD) | * Digests (Fermentas FD) | ||
** E/X | ** E/X | ||
** Inserts are gBlocks = 2.0 ng/μL | |||
{| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table --> | {| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table --> | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA ( | | DNA (20 ng) || 10.0 μL | ||
|- | |- | ||
| 10x buffer || 2.0 | | 10x buffer || 2.0 | ||
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| XbaI || 1.0 | | XbaI || 1.0 | ||
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| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 6.0 | ||
|- | |- | ||
| || 20.0 μL --> 37°C/ ~15 min. | | || 20.0 μL --> 37°C/ ~15 min. | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA (digest) || 20.0 μL ( | | DNA (digest) || 20.0 μL (20 ng) | ||
|- | |- | ||
| phosphatase || 1.0 | | phosphatase || 1.0 | ||
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| dH<sub>2</sub>O || --- | | dH<sub>2</sub>O || --- | ||
|- | |- | ||
| || 20 | | || 20 μL | ||
|} | |} | ||
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL | |||
* Ligation/ | * Digestion/Ligation Reactions (borrowed from Rene's gRNA/Cas9 assembly procedure) | ||
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector ratio | ** Inserts are gBlocks = ~2ng/μL | ||
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio | |||
* | # pAub/AubR/MRV: pAub(E/Xdp)/153 + AubR/MRV(BbsI)/4087 | ||
# pBja/BjaR/MRV: pBja(E/Xdp)/122 + BjaR/MRV(BbsI)/3977 | |||
# pRpa/RpaR/MRV: pRpa(E/Xdp)/136 + RpaR/MRV(BbsI)/3980 | |||
* Note: BraR cloning was not successful, omit from this round of assembly | |||
{| {{table}} cellspacing="3" <!-- Lig/Dig rxn table --> | {| {{table}} cellspacing="3" <!-- Lig/Dig rxn table --> | ||
| || 1 || 2 || | | || 1 || 2 || 3 | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 5.0 || 5.0 || 5.0 | ||
|- | |- | ||
| Vector DNA || | | Vector DNA || 0.2 || 0.3 || 0.2 | ||
|- | |- | ||
| | | 10x FD buf || 2.0 || 2.0 || 2.0 | ||
|- | |- | ||
| | | 10 mM DTT || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| | | 10 mM ATP || 1.0 || 1.0 || 1.0 | ||
|- | |- | ||
| || | | FD BbsI/BpiI || 1.0 || 1.0 || 1.0 | ||
|- | |||
| T4 ligase (Roche) || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 8.8 || 8.7 || 8.8 | |||
|- | |||
| || 20.0 μL || 20.0 μL || 20.0 μL || | |||
|} | |} | ||
Thermal cycler: Labnet - BbsI Dig/Lig | |||
* 6x[37°C, 5 min.; 23°C 5 min.] | |||
* 4°C, ∞ | |||
---- | |||
'''Rene - sequencing''' | |||
# g53-2, f (P139) - Confirmed | |||
# g53-2, r (P140) - low Phred 20 score | |||
# g54, f (P139) - Confirmed | |||
# g54, r (P140) - low Phred 20 score | |||
Latest revision as of 00:55, 27 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
04/23/15
Ryan - Receiver plasmids, assembly
--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL
Thermal cycler: Labnet - BbsI Dig/Lig
Rene - sequencing
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