Neon electroporation system for sirna, micro rna mimic or inhibitor transfection Reagents Required



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tarix02.03.2018
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#29244

Neon electroporation system for siRNA, micro RNA mimic or inhibitor transfection

Reagents Required:

  • Neon transfection system

  • siRNA/miRNA mimic in Rnase free water

  • PBS without Calcium and Magnesium ions - warm

  • Complete growth medium – warm

  • Table top centrifuge – at RT

  • 1.5ml, 15 ml and 50 ml tubes

  1. Harvest your cells by scraping (for RAW or BMDM) or by trypsinization ( like MEFs).

  2. Count the cell density and transfer appropriate volume to a 50 ml or 15 ml tube

  3. Centrifuge at 300-500g for 5 min at RT.

  4. During centrifuge, label 50ml tubes and add warm complete growth medium to it

  5. After centrifuge, aspirate and discard the medium

  6. Wash with 10 ml of PBS. Resuspend the pellet in PBS

  7. Centrifuge at 300-500g for 5 min at RT

  8. Aspirate PBS and discard

  9. Add an appropriate volume of R buffer to the tube and fully resuspend the pellet in R buffer. A final cell density of 1x107 can be used. Avoid storage in R buffer for longer than 30 min as it will reduce the transfection efficiency and cell viability.

  10. Transfer to 1.5 ml tubes

  11. Add the desired siRNA, micro RNA mimic or inhibitor from 40nm to 200nm depending on the optimized concentration. Mix thoroughly by pipetting up and down.

  12. Add 3 ml of E2 buffer to the Neon tube while the tube is sitting on the rack. Doing this while the Neon tube is on the Pipette station may lead to leakage of the buffer into the station and its malfunctioning.

  13. Fit the Neon tube onto the pipette station slowly until you hear the click sound. Buffer in Neon tube must be changed while switiching from one siRNA to the other or after 10 shots.

  14. Attach a Neon pipette tip to the Neon pipette

  15. Take the mixture of R buffer and siRNA/micro RNA

  16. Insert into the Neon tube on the pipette station

  17. Select the appropriate programme from the database

  18. Click on start and wait until a click sound is heard and the screen shows a complete message

  19. Transfer the electroporated sample to warm medium in 50ml tube. After final shot, mix and transfer to appropriately labelled tissue culture plates.

  20. After final use wash Neon tip and tube with sterile distilled water and store for subsequent use.

Optimized electropration protocols being used in the lab are:

RAW Voltage: 1680, Width: 20, Pulse: 1

MEF Voltage: 1050, Width: 30, Pulse: 2

BMDM Voltage: 1500, Width: 20, Pulse: 1
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