User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16: 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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==04/16/15== | ==04/16/15== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* LCR Development - PCR MV10 | * LCR Development - PCR MV10 backbone | ||
* gRNA cloning (Rene) | * gRNA cloning (Rene) | ||
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| || 50.0 || 50.0 | | || 50.0 || 50.0 | ||
|} | |} | ||
<font color=red>Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run</font> | |||
Program: Phusion (block B) | Program: Phusion (block B) | ||
* 98°C, 3 min | * 98°C, 3 min | ||
* 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, | * 35x[98°C, 10 sec; 60°C, 30 sec; 72°C, 2.5 min] | ||
* 72°C, 10 min | * 72°C, 10 min | ||
* 4°C ∞ | * 4°C ∞ | ||
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LabNet OptiMax Thermocycler | LabNet OptiMax Thermocycler: AnOlig RD | ||
* 37°C, 30 min | * 37°C, 30 min | ||
* 95°C, 5 min | * 95°C, 5 min | ||
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| bgcolor=#cfcfcf | Reagent | | bgcolor=#cfcfcf | Reagent | ||
| bgcolor=#cfcfcf | Rxn | | bgcolor=#cfcfcf | Rxn | ||
| bgcolor=#cfcfcf | Mix (x7) | |||
|- | |- | ||
| 100 ng Vector || 0.5 | | 100 ng Vector || 0.5 || 3.5 | ||
|- | |- | ||
| 1:250 dsOligo || 2.0 | | 1:250 dsOligo || 2.0 || --- | ||
|- | |- | ||
| 10x FD buf | | 10x FD buf || 2.0 || 14.0 | ||
|- | |- | ||
| 10 mM DTT || 1.0 | | 10 mM DTT || 1.0 || 7.0 | ||
|- | |- | ||
| 10 mM ATP || 1.0 | | 10 mM ATP || 1.0 || 7.0 | ||
|- | |- | ||
| FastDigest BbsI || 1.0 | | FastDigest BbsI || 1.0 || 7.0 | ||
|- | |- | ||
| | | T4 DNA ligase || 0.5 || 3.5 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 12.0 || 84.0 | ||
|- | |- | ||
| || 20.0 | | || 20.0 | ||
|} | |} | ||
LabNet OptiMax Thermocycler: BbsI Dig/Lig | |||
* 6x [37°C, 5 min; 23°C, 5 min] | * 6x [37°C, 5 min; 23°C, 5 min] | ||
* 4°C, ∞ | * 4°C, ∞ | ||
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Transformation(s) | Transformation(s) | ||
* Split ligations in 1/2, do rapid protocol for 1 set, long protocol for other set if needed | |||
* 10.0 μL ligation + 50 μL DH5α-turbo | * 10.0 μL ligation + 50 μL DH5α-turbo | ||
* Plate on 100 μg/mL amp | * Plate on 100 μg/mL amp | ||
** | |||
RESULTS (4/17/15) | |||
* Success! Plates 1-3, 5, 6 had ~5 colonies. Plate 4 had just one. | |||
* Pick two colonies from each (except plate 4, pick 1) | |||
* Make streak plate & set up 5 mL cultures | |||
Latest revision as of 00:54, 27 September 2017
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04/16/15
LCR Development
Measure conc.
Mistake: Buffer volume should be 10/rxn, no 5! Consider re-run Program: Phusion (block B)
Conclusion
gRNA cloning - Rene
Phosphorylate and anneal oligo pairs
LabNet OptiMax Thermocycler: BbsI Dig/Lig
RESULTS (4/17/15)
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