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| Line 6: |
Line 6: |
| | colspan="2"| | | | colspan="2"| |
| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| ==mm/dd/yy== | | ==01/09/13== |
| <!-- Precede each finished task with a checkmark ✓ --> | | <!-- Precede each finished task with a checkmark ✓ --> |
| * 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 5 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 ||
| |
| |-
| |
| | || total vol || --> 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°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°C overnight<br>
| |
|
| |
|
| <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Project name
