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Protocols: ES Cell Culture - Gene Targeting - Protocols

Recipes for Tissue Culture Selective Drug Stocks


100X G418 (Geneticin):  Obtained as crystalline powder from 
GIBCO (cat.# 860-1811) and stored @ room temp. in T932.

		The amount of active ingredient per total weight 
is printed on the bottle label.  (This value is generally about 
500 mg/mg, active ingredient/total weight).

		The 100X stock solution is prepared in PBS to give 
a final stock concentration of 18 mg active ingredient per ml.

Procedure:

--	Into a sterile, conical 50 ml tube, weigh an amount of 
G418 equal to the quantity required to prepare either 20 or 
40 ml of 100X solution.  
	Example:

	You need to prepare 20 ml of 100X G418.  The stock G418 
powder is 495 mg/mg active ingredient.  Perform the following 
conversion to calculate the amount of G418 to weigh out:
	-	(18 mg active ingredient/ml)  x  20 ml  =  360 mg active 
ingredient required
	-	(360 mg active)  /  (0.495 mg active/total weight)  =  727 mg 
total weight

--	Working in the hood, aseptically add enough PBS to dissolve 
the G418, but do not go over the final volume mark (the graduation
marks on the side of the tube are accurate enough for this 
determination).  The solution will be pale yellow in color (acidic).  
Once dissolved, bring to the desired volume with PBS.

--	After mixing, filter sterilize the 100X G418 through a 0.2 mm 
syringe filter, aliquot into 25 ml Nunc Universals, label with 
"100X G418" and the date, and store @ 4o C.

1000X 6TG (6-thioguanine):  Obtained as crystalline powder from 
Sigma (cat.# A-4882, listed as 2-amino-6-mercaptopurine) and stored 
@ room temp. in T932.  C5H5N5S  FW=167.2.

		The 1000X stock solution is prepared in PBS (alkalinated 
with NaOH) to give a final stock concentration of 10 mM.
	
	Procedure:

--	Into a sterile, conical 50 ml tube, weigh 1.672 mg of 6TG 
powder per ml of solution being prepared (e.g., if preparing 20 ml, 
weigh out 33.4 mg of 6TG).

--	Working in the hood, aseptically add enough PBS to dissolve 
the 6TG, but do not go over the final volume mark.  To get the 6TG 
completely in solution, add 5 N NaOH dropwise until the powder is 
fully dissolved.  The solution will be pale pink in color (alkaline).  
Once dissolved, bring to the desired volume with PBS.

--	After mixing, filter sterilize the 1000X 6TG through a 0.2 mm 
syringe filter, divide into aliquots of 0.5 ml in sterile freezing 
vials, label with "1000X 6TG", and store @ -20o C.

1000X FIAU [1-(2'-deoxy-2'-fluoro-b-d-arabinofuranosyl)-5-iodouracil]: 
	Obtained as crystalline powder from Bristol Myers (patent now owned 
by Oclassen Pharmaceuticals, Inc.) and stored in a light-proof, 
brown bottle @ 4o C in T932.  C9H10N2O5FI  FW=372.08 (molarity 
calculations based on an estimated FW of 388).

		The 1000X stock solution is prepared in PBS (alkalinated 
with NaOH) to give a final stock concentration of 200 mM.

Procedure:

--	Prepare an intermediary 100 mM stock solution by adding 388 mg 
of FIAU to 8 ml of PBS.  To get the FIAU completely in solution, add 
5 N NaOH dropwise until the powder is fully dissolved.  Once dissolved, 
bring the volume to 10 ml with PBS.  Divide into 1 ml aliquots and store 
@ -20o C.

--	When needed, prepare stocks of 1000X FIAU by adding 100 ml of 
the thawed 100 mM intermediary stock FIAU to 50 ml of PBS.  If the 
FIAU precipitates, add 5 N NaOH dropwise until the FIAU goes back 
into solution.

--	After mixing, filter sterilize the 1000X FIAU through a 0.2 mm 
syringe filter, aliquot into sterile freezing vials, label with 
"1000X FIAU", and store @ -20o C.

100X HAT Supplement (hypoxanthine-aminopterin-thymidine):
		Obtained in 10 ml lyophilized form from GIBCO 
(cat.# 320-1062AS) and stored @ 4o C, before and after rehydration.
		
Procedure:

--	Fill a 10cc syringe to the 10cc mark with Milli-Q water; 
remove any air bubbles.

--	Affix a 0.22 mm syringe filter unit to the syringe, and 
then affix a 21g 11/2" needle to the end of the syringe filter.

--	Remove the metal protective cap from the bottle of HAT 
Supplement so that the rubber stopper is exposed.

--	Pierce the rubber stopper with the needle.  The negative 
pressure in the bottle will pull the water from the syringe through 
the filter and into the bottle.

--	When all 10cc of water has been added to the bottle, remove 
the needle.  Shake the bottle to ensure complete rehydration of the 
lyophilized supplement.

--	Once completely dissolved, transfer the 100X HAT to a sterile, 
15 ml conical tube, label with "100X HAT" and the date, and store 
in the In Use tray @ 4o C in T934.

100X HT Supplement (hypoxanthine-thymidine): Obtained premixed 
in 100 ml bottles from GIBCO (cat.# 320-1067AF) and stored, 
ready to use, @ 4o C in T934.

		Because residual amounts of the aminopterin remain 
in the cells for several days after removal of HAT selection, the 
de novo pathway of purine synthesis will continue to be blocked.  
Therefore, the media must be supplemented with 1X HT for 2-4 days 
after removal of HAT selection in order for hypoxanthine and 
thymidine to be incorporated through the hprt pathway.