Biomod/2012/HKBU/BU Magician:Labook:Notebook/Labbook/2012/09/25

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Overall Plan

One set is generally the repeat of the set done on 2012/9/12-2012/9/20(Namely Plan A). Another set is a try for another method(Namely Plan B).

For Plan A: Solution Preparation

(In the following table, “*” indicates “with”, while “X” means “without”.)
HKBU2012Table6.jpg

(1) Nanoparticle solution preparation
0.01g of nanoparticle solids is dissolved with 2ml Serum Free Medium (Concentration: 5mg/ml)
Further dilution:
HKBU2012Table7.jpg
Total volume 2ml for each concentration

(2) SiRNA containing solution preparation:
For each well, 300μl of nanoparticle solution is needed.
For SiRNA containing nanoparticle solution
Total volume:
300x 2(duplicates) x2(with magnetic field/without magnetic field)=1200μl

Take in account the pipetting error, 1200μl -> 1600μl for each concentration solution containing nanoparticles

SiRNA stock concentration:40μM
SiRNA concentration needed for treatment 200nM

HKBU2012Table8.jpg
( *Remarks:The final concentration of SiRNA containing nanoparticle solution is not calculated here.)

For SiRNA containing incomplete medium solution (experiment 7,8)
Volume of SiRNA containing medium
= 300 μlx2 (duplicates) x 2(experiment7,8)
=1200 μl

Take in account of pipetting error, 1200 μl-> 1600 μl
HKBU2012Table9.jpg

(3)Conjugation
After adding SiRNA to both nanoparticle and HBSS solution, the tubes are stored in 4 centigrade refrigerator to allow conjugating overnight for 24hrs.

For Plan B: Solution Preparation & Seeding Cell

Solution preparation
(In the following table, “*” indicates “with”, while “X” means “without”.)

HKBU2012Table10.jpg

(1) Nanoparticle solution preparation
0.01g of nanoparticle solids is dissolved with 2ml incomplete medium (Concentration: 5mg/ml)
Solution with concentration 5 times as working concentration is prepared here.
HKBU2012Table11.jpg

Total volume 500 μl for each concentration


(2) SiRNA containing solution preparation:
For each well, 300μl of nanoparticle solution is needed
For SiRNA containing nanoparticle solution
60 μl is added to 240 μl serum free medium to dilute 5 times
Total volume:
60x 2(duplicates) x2(with magnetic field/without magnetic field)=240μl

Take in account the pipetting error, 240μl -> 320μl for each concentration solution containing nanoparticles

SiRNA stock concentration:40μM
SiRNA concentration needed for treatment 200nM
SiRNA concentration for nanoparticle solution preparation: 1000nM
HKBU2012Table12.jpg
( *Remarks:The final concentration of SiRNA containing nanoparticle solution is not calculated here.)
40*1000*X=1000(concentration)*320 X=8
Remarks: if under this condition, the nanoparticle is too concentrated for conjugation.

For SiRNA containing incomplete medium solution (experiment 7,8)
Volume of SiRNA containing medium
= 60μlx2 (duplicates) x 2(experiment7,8)
=240 μl

Take in account of pipetting error, 240 μl-> 320 μl
HKBU2012Table13.jpg

(3)Conjugation
After adding SiRNA to both nanoparticle and HBSS solution, the tubes are stored in 4 centigrade refrigerator to allow conjugating overnight for 24hrs.



Seeding Cells
(1) Precoat the 4 well plate
(2) Seeding cells
cell count: 714
4 well plate: 5x105cells/well
Calculate the volume of cell solution added. After cell seeding, place in incubator overnight to allow cell growth.