User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/27: 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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== | ==05/27/15== | ||
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* | * Rene - hi-res melt curve screen on CRISPR clones | ||
* | * Ben - continue luc replacement donor cloning | ||
---- | ---- | ||
''' | '''Rene - hi-res melt curve screen on CRISPR clones''' | ||
* See notes from Cold Spring Harbor: http://openwetware.org/wiki/Haynes_Lab:Notebook/CRISPR_Editing/2014/08/05 | |||
* | * Set-up | ||
** | ** 2 replicates per clone | ||
** Total wells: 36 (1 well per rxn.) | |||
** '''Template+water Multiplier''' = none (use 10x diluted stock for all rxns) | |||
** '''Primer+SYBR Mix Multiplier''' = 27 + 3 = '''39''' | |||
* Template+ Water: Dilute PCR reaction 10x | |||
** Use multichannel to transfer 5.0 μL PCR products into 45.0 μL dH<sub>2</sub>O | |||
** Set up dilutions in 8-tube strips | |||
** Use 4.5 μL Template+water per rxn | |||
# Lu34_AA01 - 12 | |||
# Ga34_AA01 - 12 | |||
# nTc (3 wells) | |||
* Master Mix: Primers+SYBR | |||
** F = P149 | |||
** R = P160 | |||
** 10.5 per well | |||
{| {{table}} | |||
| | |||
|- | |- | ||
| | | Reagent || Volume || (x30) | ||
|- | |- | ||
| | | 750 nM F/R primers || 3.0 || 90.0 | ||
|- | |- | ||
| | | 2x SYBR MM || 7.5 || 225 | ||
|- | |- | ||
| || | | || 15.0 | ||
|} | |} | ||
* | * Plate loading | ||
** Pipette 21 Primers+SYBR (2 x 10.5) into first row (A1-12) | |||
** Multichannel-aliquot 10.5 Primers+SYBR into next row (B1-12) | |||
** Pipette 10.5 Primers+SYBR into C1-3 (nTc wells) | |||
** Multi-channel pipette 4.5 Template+water into appropriate wells (A1-12, B1-12) | |||
** Pipette 4.5 water into C1-3 (nTc wells) | |||
* Run PCR - Roche LC480: "SYBR Karmella CNV melt curve Template" | |||
** 95°C, 5 min | |||
** 30x [95°C, 10 sec; 60°C, 10 sec; 72°C, 10 sec (measure)] | |||
** Melt: continuous acquisition; 95°C, 4 sec; 70°C, 60 sec, -- +0.1°C/ 5 sec (programmed as "6 acquisitions/°C") --> 95°C | |||
** Cooling: 40°C, 30 sec | |||
RESULTS: | |||
# Lu34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C | |||
# Ga34_AA01 - 12 -- Melt temps 85.1 ± 0.1 °C; additional short peak ~87°C | |||
# nTc (3 wells) -- no amplification | |||
* | * Try using more diluted template (100x) | ||
* Continue by sequencing a few purified PCR products with P215 to find a reference sample (wild type sequence) | |||
Latest revision as of 00:59, 27 September 2017
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05/27/15
Rene - hi-res melt curve screen on CRISPR clones
RESULTS:
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