Daniel Clay Austin at 23:12, 15 January 2013

Daniel Clay Austin — Tue, 15 Jan 2013 23:12:03 GMT

←Older revision		Revision as of 23:12, 15 January 2013
Line 1:		Line 1:
		+	{{Template: Sack Lab}}<br>
		+	<div style="right: center; text-align: left; float:none">
		+
	Plating subclones for fluorescent screening		Plating subclones for fluorescent screening
	For every 12 colonies picked, you will need:		For every 12 colonies picked, you will need:
Line 35:		Line 38:

	9. Freeze when T-75 is 50-90% confluent.		9. Freeze when T-75 is 50-90% confluent.
-
-
-
-	*Return to [[Sack:Protocols]]

Jon Sack: New page: Plating subclones for fluorescent screening For every 12 colonies picked, you will need: 21ml appropriate maintenance media
1 TC-treated 12-well plate
1.5 8 well chambered cove...

Jon Sack — Tue, 09 Oct 2012 01:46:22 GMT

New page: Plating subclones for fluorescent screening For every 12 colonies picked, you will need: 21ml appropriate maintenance media 1 TC-treated 12-well plate 1.5 8 well chambered cove...

New page

Plating subclones for fluorescent screening
For every 12 colonies picked, you will need:
21ml appropriate maintenance media 
1 TC-treated 12-well plate 
1.5 8 well chambered coverglass slides 
1 10ml serological pipet 
serological pipetor 
15 200 ul pipet tips (3 rows of 12) 
200 ul pipetor 
waste box for pipet tips 
pen 

1. Choose 12 wells on the 96-well plate that contain one fluorescent colony. Choose from the lowest density plate first.

2. Remove media from selected wells, rinse once with 200ul PBS, then add 50 ul trypsin. Watch to see when cells detach.

3. While cells are trypsinizing:
put 1 ml media in each well of 12-well plate
put 0.5ml media into each of the 12 chambers
mark each well and chamber with name of a chosen well from the 96 well plate

4. Once cells detach, to each well
add 200ul of media
triturate 5x to suspend cells and remove 200ul suspension
plate one drop of suspension in appropriately marked chamber
place the rest of the cells in appropriate well of 12 well plate

5. Add 200 ul media to picked wells of 96-well plate, this makes a backup from straggling cells.

6. Place cells in incubator.

7. Check chambered slides for fluorescence 1-2 days later.

8. Plate upto 6 good colonies in T-75, using all cells in the 12 well plate when confluency is 50-90%.

9. Freeze when T-75 is 50-90% confluent.

*Return to [[Sack:Protocols]]

Sack: Plating Subclones for Flourescent Screening - Revision history

Daniel Clay Austin at 23:12, 15 January 2013

Jon Sack: New page: Plating subclones for fluorescent screening For every 12 colonies picked, you will need: 21ml appropriate maintenance media 1 TC-treated 12-well plate 1.5 8 well chambered cove...

Jon Sack: New page: Plating subclones for fluorescent screening For every 12 colonies picked, you will need: 21ml appropriate maintenance media
1 TC-treated 12-well plate
1.5 8 well chambered cove...