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trh

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性别: MM
专业: 生物技术

[交流] 急求：基因的洋葱表皮细胞定位失败的原因

本人构建了一个含eGFP融合基因的植物表达载体，轰击洋葱表皮细胞后，未出现绿色荧光，请求各位有没有相似的经历，有没有没出现绿色荧光实验失败的文献可以参考，最好是有分析的失败的原因的文献，我需要查找失败的原因。必重谢，急求！

[ Last edited by 350784478 on 2009-9-19 at 12:50 ]

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lynn5180

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贴个英文的我们一直按照这个做的，如果需要电子版的PROTOCOL请M我
Preparation for the Bombardment

DNA
Prepare a Midi-Prep of your plasmid DNA and adjust to a concentration of 1µg/µl
Plant material
Order e.g. barley plants one week prior you plan to do the bombardments and let them grow in a light chamber.
Cut off the 7 days old leaves and place it on benzimidazol containing agar plates 4 hours before the bombardment and place them back into a light chamber.

Washing microcarriers (gold particles)
The following procedure prepares gold (micro carriers) for 10 bombardments. The micro carrier can be stored at 4°C for up to 2 weeks.
Weigh out 30 mg of 1m gold micro carrier into a 1.5 ml eppi.
Add 1 ml of 70% EtOH and vortex vigorously for 3 - 5 minutes on a platform vortexer.
Allow the particles to sink for 15 minutes.
Pellet the micro particles by spinning for 5 seconds at RT.
Discard the supernatant

Repeat the following wash step three times :

Add 1 ml of sterile water.
Vortex for 1 minute
Allow the particles to settle for 1 minute.
Pellet the micro particles by spinning for 5 seconds
Remove the liquid and discard.

Add 500 l sterilized 50%glycerol / 50% water to adjust the micro particle concentration to 60 mg/ml

Coating micro carriers with DNA
The following procedure prepares one bombardment. Coated micro carriers should be prepared 1 hour before using.

Vortex the micro carrier prepared in 1:1 water/Glycerol for 5 minutes on a platform vortexer to resuspend and disrupt agglutinated particles.
Remove 50 l of micro carriers while vortexing  and add to a 1.5 ml eppi.
While vortexing vigorously, add in order:
   5 l DNA  (1g / l)
50 l    2,5 M CaCl2
20 l 0,1 M Spermidine
Continue vortexing for 2 - 3 minutes.
Allow the micro carriers to settle for 1 minute.
Pellet micro carriers by spinning for 2 seconds.
Discard the liquid.
Add 140 l 70% EtOH.
Vortex at low speed for 2 seconds, spin for 2 seconds.
Remove the liquid and discard.
Add 140 l 100% EtOH.
Vortex at low speed for 2 seconds, spin for 2 seconds.
Remove the liquid and discard.
Resuspend the coated gold particles in  60 l 100% EtOH.
Keep on ice until using for bombardment.
Bombardment
Turn on the PDS1000/HE and the vacuum pump
The first bombardment each day should be a "dry run" with no micro carrier to ensure that the system is set up properly and the gas pathway is filled with helium.
(Procedure see below, but without samples)
Each of seven macro carrier is placed inside a macro carrier holder, and fixed with the plastic insertion tool
For each macro carrier, remove 7 l aliquots of micro carriers, pipet from a continuously vortexed tube and spread it on the macro carrier using a pipet tip
The ethanol should evaporate completely
Briefly wet the rupture disk in 2-propanol
Turn the MC-holder upside down and intercalate the SS Holder with a clean Stopping Screen directly below the browser (platform2).
Align the browser exactly above the macro carrier.
Place the target shelf with the placed sample at the desired level inside the bombardment chamber (platform4).
Close and latch the sample chamber door.
Turn on the vacuum source
Set the vacuum switch to the VAC position until 27 inches of mercury is reached and hold this level by quickly pressing the vacuum control switch through the middle VENT position to the bottom HOLD position.
With the vacuum level in the bombardment chamber stabilized, press and hold the FIRE switch to allow the helium pressure inside the gas acceleration tube that is sealed by the selected rupture disc and observe the helium pressure gauge at the top of the acceleration tube, until the rupture disc bursts.
Release the vacuum in the sample chamber by setting the VAC switch to the middle VENT position.
Open the sample chamber door when the vacuum gauge reaches 0 inches of mercury of vacuum.

After bombardment pick up the leaves, place it back on the petri dish

2.5 M CaCl2 stock solution
Autoclave

0.1 M Spermidine
(N-[3-Aminopropyl]-1,4butanediamine)
SIGMA Order No.: S-4139,1g
Dissolve 1 g spermidine with 67,80 ml water
Sterilize by filtration
Aliquot and store at -20°C

50% glycerol / 50% water (v/v)
Autoclave

1% Agar, 85 M benzimidazol plates

100 x benzimidazol stock
Sterile filter

Rupture Disks, 900 psi
Inbio Gold
Cat.No.: BD900
Microcarreier
BioRad
Gold Particles, 1,0 micron
Cat.No.: 1652225
Macrocarrier
Inbio Gold
Macrocarrier
Cat.No.:BD042

[ Last edited by lynn5180 on 2009-6-10 at 22:19 ]