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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<!-- 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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| 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) | ||
** Inserts are gBlocks = ~2ng/μL | ** Inserts are gBlocks = ~2ng/μL | ||
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio | ** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio | ||
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| Vector DNA || 0.2 || 0.3 || 0.2 | | Vector DNA || 0.2 || 0.3 || 0.2 | ||
|- | |- | ||
| 10x buf | | 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: | Thermal cycler: Labnet - BbsI Dig/Lig | ||
* | * 6x[37°C, 5 min.; 23°C 5 min.] | ||
* 4°C, ∞ | * 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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