Biology Reference
In-Depth Information
15 min at 121 °C. Prior to pour plates, melt the LB-Agar in
a microwave (approx. 500 W) and add 1/1,000 volume
ampicillin or carbenicillin (end concentration 100
g/mL)
just before the solution is lukewarm. Pour about 20 plates
(storage at 4 °C in the dark).
14. Stbl3 E. coli strain is used for transformation (
see
Note 4
).
Stbl3 genotype: F−
mcr
B
mrr hsd
S20(rB−, mB−)
rec
A13
sup
E44
ara
-14
gal
K2
lac
Y1
pro
A2
rps
L20(StrR)
xyl
-5
μ
λ
−
leu mtl
-1.
15. PC8 (Phenol-Chloroform, pH 8).
16. 3 M Sodium acetate, pH 5.2: Dissolve 40.82 g sodium acetate-
trihydrate in 80 mL water. Adjust to pH 5.2 with glacial acetic
acid and fi ll volume up to 100 mL.
17. Glycogen 20 mg/mL (e.g., Roche).
18. Primers:
-pLKO-forward: 5
′
-GACTGTAAACACAAAGATATTAG-3
′
.
-pLKO-reverse: 5
′
-TAATTCTTTAGTTTGTATGTCTG-3
′
.
.
19. 10× RDA buffer: 670 mM Tris (pH 8.8), 160 mM ammonium
sulfate, 100 mM
-pLJM1-forward: 5
′
-TAACAACTCCGCCCCATTGA-3
′
-mercaptoethanol, 1 mg/mL BSA.
20. 50 mM MgCl
2
: Dissolve 1.02 g MgCl
2
× 6H
2
O in 100 mL.
21. Midi-Kit for purifying vectors after cloning, e.g., PureYield™
DNA Midiprep System (Promega).
22. Glycerol, 99.5 %.
β
2.3 Lentiviral
Particles
1. 0.1 M NaPO
4
solution (pH 7.1): Dissolve 2.76 g NaH
2
PO
4
-
1H
2
O in 100 mL H
2
O. Dissolve 5.36 g Na
2
HPO
4
-7H
2
O in
100 mL H
2
O. Mix both solutions (~1:2) to obtain pH 7.1 and
double the volume with water.
2. 2× HBS buffer (HEPES buffered saline): 50 mM HEPES,
280 mM NaCl, 1.5 mM NaHPO
4.
Dissolve 6.5 g HEPES, 8 g
NaCl, and 7.5 mL 0.1 M NaPO
4
(pH 7.1) in 450 mL H
2
O.
Adjust pH exactly to 7.1, bring up to 500 mL with H
2
O and
aliquot in 50 mL tubes.
3. 2 M CaCl
2
: 87.6 g CaCl
2
-6×H
2
O in a fi nal volume of 200 mL
water. Filter sterilize and store at 4 °C.
4. Polybrene (Hexadimthrine bromide): 4 mg/mL solution in
water. The aliquot in use can be stored at 4 °C.
5. DMEM supplemented with 10 % fetal calf serum, 100 U/mL
penicillin, and 100
g/mL streptomycin.
6. Puromycin: Dissolve puromycin in water at 50 mg/mL and
store at −20 °C in aliquots.
7. Phosphate-buffered saline (1× PBS, pH 7.4): commercial
source.
μ
