User:Karmella Haynes/Notebook/Polycomb project/2013/01/09: 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"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|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]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==mm/dd/yy==
==01/09/13==
<!-- Precede each finished task with a checkmark &#x2713; -->
<!-- Precede each finished task with a checkmark &#x2713; -->
* Outline of experiments/ tasks
* Thaw new cells: U2OS Flp-in TRex, HEK293 Flp-in TRex


----
----
'''Task 1'''<br>
'''Thaw cells'''<br>
> Line item 1<br>
* Quick thaw. Add to 7 mL FBS-tet-free
 
* Spin for 3 min., 1000 rpm, r.t.
{| class="wikitable" border="0" cellspacing="5"
** U2OS Flp-in TRex - reuspend in McCoy's 5A/ 10% FBS/ 1% p/s
|-
** HEK293 Flp-in TRex - reuspend in DMEM / 10% FBS/ 1% p/s
| <u>Reagent</u>  || <u>Volume</u> || <u>Conc.</u>
* Grow in small flat bottom flasks
|-
| reagent 1      || vol 1 || conc 1
|-
| reagent 2      || vol 2 || conc 2
|-
| dH<sub>2</sub>O || vol 3 || &nbsp;
|-
| &nbsp;          || total vol || &nbsp; --> Reaction conditions
|}
 
----
'''Lipo transfection'''<br>
> Details
 
{| class="wikitable" border="0" cellspacing="5" <!-- Lipofectamine transfection table -->
|-
| <u>Wells</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Lipo</u>  || <u>Opti-MEM (total)</u>
|-
| 1            || name          || 2 μg      || ### μL        || 4 μL          || 500 μL
|-
| 2            || name          || "          || ### μL        || "            || "
|-
| 3            || name          || "          || ### μL        || "            || "
|-
| 4            || name          || "          || ### μL        || "            || "
|-
| 5            || name          || "          || ### μL        || "            || "
|-
| 6            || name          || "          || ### μL        || "            || "
|}
 
> Add Lipo to 250 μL Opti-MEM --> R.T/ 5 min. <br>
> Add DNA to 250 μL Opti-MEM <br>
> Add DNA mix to Lipo mix --> R.T./ 20 min. <br>
> '''Remove 500 μL medium from each well'''; Add 500 μL complexes to each well (4 ml med. each); Grow cells at 37&deg;C<br>
> Refresh medium after 5 hours (ab-free)<br>
 
----
'''Fugene transfection'''<br>
> Details<br>
 
{| class="wikitable" border="0" cellspacing="5" <!-- Fugene transfection table -->
|-
| <u>Wells</u> || <u>Plasmid</u> || <u>DNA</u> || <u>Volume</u> || <u>Fugene</u> || <u>Opti-MEM</u>
|-
| 1-6          || name          || 200 ng    || ### μL        || 1.8 μL        || 18.2 μL
|-
| 7-12        || name          || "          || ### μL        || "            || "
|-
| 13-18        || name          || "          || ### μL        || "            || "
|-
| 19-24        || name          || "          || ### μL        || "            || "
|}
 
> Master mixes (4, x6): 10.8 μL Fugene + 109.2 μL Opti-MEM --> R.T/ 5 min. <br>
> Add 6x vol DNA --> R.T./ 20 min. <br>
> Add 20 μL complexes to each well (1 ml med. each); Grow cells at 37&deg;C overnight<br>


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Latest revision as of 22:20, 26 September 2017

Project name Main project page
Next entry

01/09/13

  • Thaw new cells: U2OS Flp-in TRex, HEK293 Flp-in TRex

Thaw cells

  • Quick thaw. Add to 7 mL FBS-tet-free
  • Spin for 3 min., 1000 rpm, r.t.
    • U2OS Flp-in TRex - reuspend in McCoy's 5A/ 10% FBS/ 1% p/s
    • HEK293 Flp-in TRex - reuspend in DMEM / 10% FBS/ 1% p/s
  • Grow in small flat bottom flasks


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