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- 积分
- 981
- 威望
- 981
- 包包
- 1968
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Protocol for virus production in plat-E cells
, p5 A3 \) n VPrepare plat-E cells for transfection
. J! a6 h# F* B+ l4 L- r9 }9 F" gThe evening before transformation, plate plat-E cells at 1.3-1.35x106 cells/6-well cell culture plate(8×106cells for each 100 mm plate).
) H5 l5 m# @% X+ ?Transfection with plasmid mix using CaPO4 precipitation
. z5 [: s# N+ @3 }1.Observe the plates seeded the day before. When cells are about 80% confluence they are ready for transfection. Change the medium with pre warmed complete medium 1-2hrs before transfection(1.5 mL medium for each well of 6-well plate;while 7.5 mL medium for each 100 mm plate.).3 n$ W2 r! u+ S, o4 V. i
2. Preparation of Vector Mixes for transfection in plat-E Cells at a ratio below(for 6-well cell culture plate):0 r7 M0 t8 C( z4 F5 {3 M; [
Stocking concentration Working concentration Each well for 6-well plate for 100mm plate
/ x. d8 N- ~5 p& B cDNA(GFP) 0.7 μg/μL 10 μg/mL 7.14 μL 35.72 μL
: A: z& }0 s% Z! A2 k- G' w) FH2O / / 211.61 μL 1058.03 μL
* h0 c$ W, ]' ], T, KCaCl2 2 M 125 mM 31.25 μL 156.25 μL
4 }6 w" `+ W6 M! T' P4 H1 a2×HBS 2× 1× 250 μL 1250 μL
. b" l. v" C& _: ~total / / 500 μL 2500 μL2 h# z' X" D5 [. R1 ~; V
1)Dilute the DNA with ddH2O,mix well.
' K) A, G) d3 R3 d5 T6 B2)Add 31.25μL 2M CaCl2 to DNA solution,mix well.7 G. W: W0 g2 a2 B
3)Add 250μL 2×HBS to the CaCl2/DNA mixture, pipet 30 times to make sure the solution is mixed well.
* B! @3 s& g0 N; [2 R4 m: s l! S3. Incubate the DNA/calcium phosphate mixture at room temperature for 2min.And then add this mixture to the cells gently,drop by drop. Mix well,until the medium has recovered a uniformly red color. Make sure you evenly sprinkle the droplet over the entire area.0 _6 w2 p& [9 ]& X: N
4. Incubate the cells at 5%CO2, 37 °C overnight (11–14 h). ( E. j1 {/ D- L+ U; v
5. Next morning(about 11h after transfection), gently remove medium, and very gently replace with 2ml fresh medium for each well(10 mL for each 100 mm cell culture plate). Incubate at 5%CO2, 37 °C.5 F. h, F$ I5 o
6.Harvest virus at appropriate time.2 f8 Z) ]; Y& X+ _. Z# G/ N6 F
Reagents and solutions
& ^2 R. S! P6 p7 j; m2 j4 T% o/ ?9 bDNA: The plasmid DNA to be used in the transfection should be free of protein, RNA and chemical contamination.A DNA solution with an A260/A280 ratio of 1.8 or greater is desirable.
2 w4 y% `, j! n" U) u& IH2O:Millipore water, filter sterilize through 0.22-μm nitrocellulose filter.& w$ J h! M/ M8 m# S
2M CaCl2(SigmaUltra): filter sterilize through 0.22-μm nitrocellulose filter.
( z1 _- l8 U) i! h$ s9 v6 a# D% {* o2×HBS:$ z) w8 x8 G! V
280 mM NaCl(SigmaUltra); X' x7 ^+ F4 W+ b+ R* d* L& q
10 mM KCl(SigmaUltra). u/ H2 R6 S% D9 B# w$ c
1.5mM Na2HPO4(SigmaUltra)
& x! S# B5 a& l! F0 D6 D9 A8 D50mM Hepes(SigmaUltra)) M" s& J$ y5 i; O' J) @
12mM Glucose(SigmaUltra)
0 L5 |0 `$ w" a5 ?. k1 ^/ v* O; afilter sterilize through 0.22-μm nitrocellulose filter.
3 w7 u) c2 j* c/ u5 N# }这是我们用的protocol，HBS的PH也是配几个不同的测试选最好的，11-14小时换液， 注意转染前细胞的密度，漂很有可能是细胞太多了 |
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总评分: 威望 + 5
包包 + 15
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发表于 2010-8-7 11:37
发表于 2010-8-20 10:37
