Haynes Lab:Notebook/CRISPR Editing/2015/05/22: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</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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'''SYBR qPCR to determine plasmid counts for dilution experiment''' | '''SYBR qPCR to determine plasmid counts for dilution experiment''' | ||
{| {{table}} | {| {{table}} | ||
| pX330 primer|| | | pX330 primer||P209/210 (G) | ||
|- | |- | ||
| internal primer|| | | internal primer||18S (C) | ||
|- | |- | ||
| Dilution||2ng/rxn | | Dilution||2ng/rxn | ||
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| align="center" style="background:#f0f0f0;"|'''rep''' | | align="center" style="background:#f0f0f0;"|'''rep''' | ||
| align="center" style="background:#f0f0f0;"|'''ng/µL''' | | align="center" style="background:#f0f0f0;"|'''ng/µL''' | ||
| align="center" style="background:#f0f0f0;"|'''dilute 1: | | align="center" style="background:#f0f0f0;"|'''dilute 1:20''' | ||
| align="center" style="background:#f0f0f0;"|'''ul dilution''' | | align="center" style="background:#f0f0f0;"|'''ul dilution''' | ||
| align="center" style="background:#f0f0f0;"|'''ul water''' | | align="center" style="background:#f0f0f0;"|'''ul water''' | ||
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:{| {{table}} | :{| {{table}} | ||
| ||1-3||4-6||7-9||10-12 | | align="center" style="background:#f0f0f0;"|'''''' | ||
| align="center" style="background:#f0f0f0;"|'''1-3''' | |||
| align="center" style="background:#f0f0f0;"|'''4-6''' | |||
| align="center" style="background:#f0f0f0;"|'''7-9''' | |||
| align="center" style="background:#f0f0f0;"|'''10-12''' | |||
|- | |- | ||
| A|| | | A||__1 G__||__1 C__||blank-G||______ | ||
|- | |- | ||
| B|| | | B||__2 G__||__2 C__||blank-C||______ | ||
|- | |- | ||
| C|| | | C||__3 G__||__3 C__||______||______ | ||
|- | |- | ||
| D|| | | D||__4 G__||__4 C__||______||______ | ||
|- | |- | ||
| E|| | | E||__5 G__||__5 C__||______||______ | ||
|- | |- | ||
| F|| | | F||__6 G__||__6 C__||______||______ | ||
|- | |- | ||
| G|| | | G||__7 G__||__7 C__||______||______ | ||
|- | |- | ||
| H|| | | H||__8 G__||__8 C__||______||______ | ||
|} | |} | ||
<br><br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Cell type''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid 1''' | |||
| align="center" style="background:#f0f0f0;"|'''Plasmid 2''' | |||
| align="center" style="background:#f0f0f0;"|'''Rep''' | |||
| align="center" style="background:#f0f0f0;"|'''260''' | |||
| align="center" style="background:#f0f0f0;"|'''280''' | |||
| align="center" style="background:#f0f0f0;"|'''260/280''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/µL''' | |||
| align="center" style="background:#f0f0f0;"|'''1:20 dil''' | |||
| align="center" style="background:#f0f0f0;"|'''ul dil for 7ng''' | |||
| align="center" style="background:#f0f0f0;"|'''ul water''' | |||
|- | |||
| Luc14||KAH226||g034||1||0.081||0.04||2.021||80.531||4.02655||1.738460965||14.01153903 | |||
|- | |||
| Luc14||KAH226||g034||2||0.126||0.061||2.06||126.451||6.32255||1.107148223||14.64285178 | |||
|- | |||
| Luc14||KAH226||g034||3||0.091||0.044||2.045||90.862||4.5431||1.540798133||14.20920187 | |||
|- | |||
| Luc14||GFP||g034||1||0.104||0.051||2.052||104.239||5.21195||1.343067374||14.40693263 | |||
|- | |||
| Luc14||GFP||g034||2||0.065||0.031||2.094||64.965||3.24825||2.155006542||13.59499346 | |||
|- | |||
| Luc14||GFP||g034||3||0.039||0.02||1.99||39.096||1.9548||3.580928995||12.169071 | |||
|- | |||
| ||||||||||||||||||||15.75 | |||
|} | |||
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05/22/2015SYBR qPCR to determine plasmid counts for dilution experiment
Step 1: make two PCR tubes for each sample with the following volumes of water and gDNA for 2ng of gDNA per well
Step 2: make primer master mixes. Will have to add 3.5x to each triplicate gDNA tube so can calculate mastermixes from that
Step 3: mix gDNA and primer mixes
Step 4: pipette plate
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