| Cepko/Tabin Lab ,Harvard University
http://axon.med.harvard.edu/~cepko/protocol/mike/V1.html
Phoenix Transfection/Viral Production (A)
This transient transfection protocol for the ecotropic/amphotropic producer line from Gary Nolan includes instructions for titering and concentration of viral stocks. I have found that this procedure gives consistently higher transfection efficiency than the protocol from the Nolan Lab.
Solutions
HN Buffer
50 mM HEPES 47.64 g HEPES (Sigma #H9136)
280 mM NaCl 65.45 g NaCl
pH to 7.1-7.3 with approximately 8 ml
10N NaOH, bring up to 4 liters with Q
and autoclave
0.5M CaCl2
29.4 g CaCl 2 (Mallinckrodt #4160)
up to 400 ml with Q
filter sterilize and store in aliquotes at -20°
Phosphate Buffer
70 mM sodium phosphate 0.966 g NaH2PO4-H2O
up to 100 ml with Q, autoclave
Carrier DNA
high molecular weight calf thymus DNA
Boehringer Mannheim #104-167
bring up to 1 mg/ml with sterile Q
store at 4°, do not freeze
50 mM Chloroquine
2.57 g chloroquine (Sigma #C6628)
up to 100 ml with Q
filter sterilize and store at -20°
1000X Polybrene
50 mg polybrene (Sigma #H9268)
up to 10 ml with Q
filter sterilize and store at -20°
1000X Diptheria Toxin
5 mg diptheria toxin (Sigma)
up to 5 ml with 1X PBS
filter sterilize and store at -20°
1000X Hygromycin
1 g hygromycin (Calibochem)
up to 3.3 ml with 1X PBS
filter sterilize and store at -20°
10X PBS
80 g NaCl
2 g KCl
14.4 g Na2HPO4
2.4 g KH 2PO4
up to 1 liter with Q, pH to 7.4 and autoclave
20% Paraformaldehyde/4% Paraformaldehyde-PBS
200 g paraformaldehyde
1 ml 10N NaOH
up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°
mix 100 ml 20% paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q
filter, and store at 4° for up to 2 weeks
0.5% Gluteraldehyde-PBS
(Nolan says to use 0.05% gluteraldehyde to fix 3T3 for titering)
0.5 ml 25% gluteraldehyde
25 ml 1X PBS
use immediately
AP Detection Buffer
(for every 7.5 ml add 25 microliters NBT and 19 microliters BCIP)
20 ml 5M NaCl
50 ml 2M Tris 9.5
50 ml 1M MgCl2
up to 1 liter with Q
store at room temperature
BCIP
0.5 g BCIP (p-toluine salt Sigma #B-8503)
up to 10 ml with DMF
store at -20° in light proof glass container
NBT
0.75 g NBT (Sigma #N-6639)
up to 10 ml with 70% DMF
store at -20° in light proof glass container
X-Gal Stain Solution
14.7 g Potassium Ferrocyanide 35 mM (Sigma #P-9387)
11.5 g Potassium Ferricyanide 35 mM (Sigma #P-8131)
0.41 g (2 mM) MgCl 2
0.02% NP40 (diluted from a 10% stock)
0.01% Deoxycholic Acid Sodium Salt (Sigma #D-6750)
50 ml 10X PBS
up to 500 ml with Q
store at 4°C in a light proof glass bottle
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