Haynes Lab:Notebook/Jan/2015/03/30: Difference between revisions
Jan Simper (talk | contribs) (Autocreate 2015/03/30 Entry for Haynes_Lab:Notebook/Jan) |
Jan Simper (talk | contribs) |
||
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. ##### --> | ||
== | ==Tissue Culture Training== | ||
* | * Passaging - U2OS, split PS3 --> PS4 | ||
6-well plate - U2OS | |||
Passaging - U2OS | |||
Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells] | |||
Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S | |||
Use 90-100% confluent T-75 | |||
Make 1 new T-75 at a 1:10 dilution | |||
Passage number: 4 | |||
6-well plate - U2OS | |||
Use same resuspension from old flask in Passaging - U2OS | |||
Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration | |||
Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium | |||
Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well | |||
Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well | |||
Make a batch of diluted cells for new 6-well plate: | |||
Into a sterile 50 mL conical add... | |||
(6 wells + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells | |||
(6 wells + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium | |||
* Passaging - KAH126, split PS3 --> PS4 | |||
6-well plate - KAH126 | |||
Passaging - KAH126 | |||
Protocol: [http://openwetware.org/wiki/Haynes:SplittingCells Splitting Cells] | |||
Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S | |||
Use 90-100% confluent T-75 | |||
Make 1 new T-75 at a 1:10 dilution | |||
Passage number: 4 | |||
Dox induction - KAH126 | |||
Induce cells in 6-well plate with dox to express PcTF (RFP) | |||
Stock concentration of dox = 1.0 mg/mL | |||
Desired final concentration of dox = 1.0 μg/mL (1000x diluted) | |||
Therefore add 1.0 μL of stock dox for every 1.0 mL medium in well | |||
4.0 mL medium in well x 1.0 μL stock dox = 4.0 μL stock dox to be added to each well | |||
Revision as of 10:11, 30 March 2015
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Tissue Culture Training
6-well plate - U2OS Passaging - U2OS Protocol: Splitting Cells Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S Use 90-100% confluent T-75 Make 1 new T-75 at a 1:10 dilution Passage number: 4 6-well plate - U2OS Use same resuspension from old flask in Passaging - U2OS Starting with: 10E6/10 mL = 1.0E6/1 mL stock cell resuspension concentration Desired cells/ well in new 6-well plate = 2.5E5 (250k) cells in 4.0 mL medium Amount of starting culture(x) to use: x = (1.0E6 cells / 1 mL) / 2.5E5 cells = 0.25 mL stock cell resuspension per well Amount of fresh medium to use: 4.0 mL final volume - 0.25 mL stock cells = 3.75 mL fresh medium per well Make a batch of diluted cells for new 6-well plate: Into a sterile 50 mL conical add... (6 wells + 1 pipetting error) x 0.25 mL stock cells = 1.75 mL stock cells (6 wells + 1 pipetting error) x 3.75 mL fresh medium = 26.25 mL fresh medium
6-well plate - KAH126 Passaging - KAH126 Protocol: Splitting Cells Cell culture medium is: McCoy's 5A, 10% FBS (tet-free), 1% P/S Use 90-100% confluent T-75 Make 1 new T-75 at a 1:10 dilution Passage number: 4 Dox induction - KAH126 Induce cells in 6-well plate with dox to express PcTF (RFP) Stock concentration of dox = 1.0 mg/mL Desired final concentration of dox = 1.0 μg/mL (1000x diluted) Therefore add 1.0 μL of stock dox for every 1.0 mL medium in well 4.0 mL medium in well x 1.0 μL stock dox = 4.0 μL stock dox to be added to each well
|