Target Discovery and Validation Reviews and Protocols

Keratin Transgenics and Knockouts 231
6. 20 mL 2X Freezing medium: mix 10 mL of complete ES medium, 4 mL of ultrapure
dimethyl sulfoxide, and 6 mL of FCS. Store at –20°C.
7. 10X HEPES-buffered saline (HBS) for electroporation: mix 16 g of NaCl, 0.74 g
of KCl, 0.252 g of Na 2 HPO 4 , 2 g of D-glucose (dextrose), and 10 g of HEPES.
Dissolve all in 180 mL of cell culture grade water, adjust pH to 7.2, bring up to
200 mL, and then filter through 0.1-µm pore filter. Store at –20°C.
To prepare 1X HBS, dilute with sterile water, which brings pH to 7.05. HAT
and HT media are bought as 50X stocks from Sigma and used as if they were
100X concentrated. To prepare 100X HAT, dissolve hypoxanthine in 2 mL of
1 N NaOH, add 8 mL of cell culture grade water, and mix well. Add another
10 mL of water and mix until most of the hypoxanthine is dissolved. Add 1 mL
of 1 N NaOH and mix until hypoxanthin is dissolved. Subsequently, add 75 mL
of water, thymidine, and then 100 mL of water. Add aminopterin and mix again
(aminopterin is a poison, so consult material safety data sheet). Filter through
0.1-µm filter and then store as 20-mL aliquots at –20°C.
8. 1 mM Aminopterin: weigh out 100 mg and dissolve in 45 mL of cell culture grade
water. Add about 1 mL of 1 N NaOH to dissolve aminopterin, sterilize as in item
7, and then store at –20°C.
9. Additional reagents:
a. 1X Trypsin inhibitor from Glycine max (soybean; cat. no. 074K1366, Sigma).
Store as 10-mL aliquots at –20°C.
b. 200X Pen/strep (Invitrogen) and stored as 1-mL aliquots at –20°C.
c. G418 (Invitrogen or any other manufacturer). Prepare 40 mg/mL in PBS and
filter sterilize. Store at –20°C. Final concentration used is 350–400 µg/mL.
d. Puromycin dihydrochloride (Calbiochem). Reconstituted in water to get a
stock concentration of 3 mg/mL. Final concentration used is 3 µg/mL.
e. Hygromycin B solution (Calbiochem). The final concentration used is 150 µg/mL.
f. Primocin (cat. no. Amaxa Biosystems) and used against mycoplasms as recommended
by supplier. When using primocin, Pen/Strep is not additionally
required in the medium.
g. 6-TG is prepared as a 1000X stock (i.e., 2 mM). Dissolve 50 mg in small volume
of 1 N NaOH. Add water up to 25 mL, dissolve completely, and filter sterilize.
Store as 5-mL aliquots at –20°C.
h. LIF is prepared according to protocol.
i. 0.3 M Mannitol (cat. no. M4125, SIgma) is dissolved in ultrapure water with
0.3% BSA added (cat. no. A4378, Sigma); filter through 0.22-µm Millipore filter
and store in aliquots at –20°C.
2.2. Websites, Tables, Vendors, and Vectors
2.2.1. Online Databases
1. http://www.mshri.on.ca/nagy/Cre-pub.html.
2. http://jaxmice.jax.org/models/cre_intro.html.