Biology Reference
In-Depth Information
6. 10 % glycerol: add molecular biology grade glycerol to deion-
ized water to fi nal concentration of 10 % (w/v) and autoclave.
Cool down on ice for at least 3 h, if it will be used for prepara-
tion of competent cells (
see
Subheading
3.4
).
7. Galactose stock solution: add
D
-galactose to deionized water
to fi nal concentration of 10 % (w/v) and autoclave.
8. 2-Deoxy-galactose (DOG) stock solution: add 2-deoxy-
galactose to deionized water to fi nal concentration of 10 %
(w/v). Sterilize by fi ltration.
9. MacConkey plates: Add 10 g MacConkey agar (Difco) to
225 mL deionized water and autoclave. Transfer the hot agar
solution into the 50 °C water bath. After reaching 50 °C, add
25 mL galactose stock solution. Mix gently and add antibiot-
ics, if required. Mix again and poor plates using ~20 mL
medium for each.
10. DOG plates Mix 4 g bacteriological grade agar (Difco) to
200 mL deionized water and autoclave. Transfer the hot agar
solution into the 50 °C water bath. After reaching 50 °C, add
50 mL 5× M63 salts, 0.5 mL MgSO
4
stock solution, 0.25 mL
D
-biotin stock solution, 1.1 mL
L
-leucine stock solution, 5 mL
10 % glycerol, and 5 mL DOG stock solution. Mix gently and
fi nally add antibiotics, if required. Mix again and poor plates
using ~20 mL medium for each.
11. For washing competent cells use molecular biology grade
deionized water, which is sterilized by autoclaving.
12.
E. coli
strain SW102 is available from Neel Copeland's labora-
tory (inquire through
http://recombineering.ncifcrf.gov
).
1. All molecular biological techniques and tools needed for these
protocols are standard and should be carried out according to
Sambrook and Russell [
14
].
2. For PCR reactions, use HPLC purifi ed primers and high
fi delity polymerases.
3. BAC DNA preparation should be carried out by advanced
column purifi cation kits according to the manufacturer's
instructions.
4. Dulbecco's Modifi ed Eagle's Medium (DMEM) (Invitrogen)
supplemented with 10 % fetal bovine serum (FCS) is used for
culturing of 293 cells.
5. For transfection of activated Ad-BACs into 293 cells,
Lipofectamine 2000 (Invitrogen) or equivalent transfection
reagents should be used according to the manufacturer's
instructions.
2.2 Molecular
Biology and Cell
Culture
