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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
- V) D7 }/ h/ A L! K5 E P! N1 qPrepare plat-E cells for transfection* v+ `% n8 X }4 P& A9 U
The 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).
/ k9 C# L2 T& l/ `% ], g0 _( STransfection with plasmid mix using CaPO4 precipitation8 a( e4 R: I6 {, @
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.).
! U' i7 r+ h" D5 P; ^! C9 S2. Preparation of Vector Mixes for transfection in plat-E Cells at a ratio below(for 6-well cell culture plate):: p# d4 W1 B3 o; B% i9 \
Stocking concentration Working concentration Each well for 6-well plate for 100mm plate) m: T8 Q4 G9 y L% J9 o( b4 G# j
DNA(GFP) 0.7 μg/μL 10 μg/mL 7.14 μL 35.72 μL! c6 ?, O" f- K2 g3 b
H2O / / 211.61 μL 1058.03 μL
7 s t; J+ X3 \7 L+ k$ ECaCl2 2 M 125 mM 31.25 μL 156.25 μL
7 N8 y+ O/ l2 d% E: j# k2×HBS 2× 1× 250 μL 1250 μL
" L& D( h! d% y/ J+ w2 W% Ktotal / / 500 μL 2500 μL$ n* Q8 [1 I* S" @7 u; P- O& v
1)Dilute the DNA with ddH2O,mix well.4 H6 T! m4 G4 }) w) r/ U b/ v
2)Add 31.25μL 2M CaCl2 to DNA solution,mix well.
o0 K% t4 z4 s5 V" q3)Add 250μL 2×HBS to the CaCl2/DNA mixture, pipet 30 times to make sure the solution is mixed well.2 p) [1 P9 t: E# |1 k9 _- m
3. 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.
$ R7 x5 b$ b% o$ O5 f% B( g# R* w& o4. Incubate the cells at 5%CO2, 37 °C overnight (11–14 h).
! |* g. E- C& w6 G; w; n5. 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.
0 V* y3 x, Z. T$ B; @6.Harvest virus at appropriate time. E$ o7 P0 X" j6 _4 n* r
Reagents and solutions
$ ^( q5 {! U' S+ zDNA: 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.
) ~) C: k+ G& v/ J" ]6 LH2O:Millipore water, filter sterilize through 0.22-μm nitrocellulose filter.7 j# w0 j7 v2 L
2M CaCl2(SigmaUltra): filter sterilize through 0.22-μm nitrocellulose filter.
6 y; a8 i; H% H5 U1 I' _: S2×HBS:- s. N0 z; n8 ~8 x" N1 {
280 mM NaCl(SigmaUltra)
* x5 x3 \" d4 k10 mM KCl(SigmaUltra)3 ~9 a; j# d f* K- t& J
1.5mM Na2HPO4(SigmaUltra)
) ~! ~( ?; |+ @# u50mM Hepes(SigmaUltra)
( z& b4 H$ D/ B! o6 K! ~' K7 `12mM Glucose(SigmaUltra)% }9 M3 [5 [5 b. k% I+ \
filter sterilize through 0.22-μm nitrocellulose filter.
2 [: z" ~9 ~2 @5 Y2 u/ I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
