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Electroporation (sorry for the repetition, i earlier posted it in the student fo


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#1 Steve Leumi

Steve Leumi

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Posted 17 April 2018 - 01:47 AM

Hi all,

 

I am Steve Leumi and I am making my first steps in the field of Microbiology; here below is my concern:

 

Two days ago, I have conducted an electroporation experiment of Huh 7.5.1 cell with HCV in-vitro transcribed RNA exactly according to the following steps.

 

1) In a 15 cm dish (around 90/100 confluent of Huh7.5.1 cells), I washed the cells with PBS and detached them with 500ml of trypsin, i then incubated them for about 5minutes at room temperature (under the hood of course),

 

2) I neutralized the trypsin using 500ml of complete medium (GIBCO)

 

3) I reproduced steps 1 and 2 in another 15cm petri dish (because i needed 20 million cells for my experiement) 

 

4) I counted the cells using the hemocytometer and I collected 20 million of cells (from the two dishes)

NB: while counting the cells, I put the trypsinized cells inside the incubator (37 degree Celcius); and the counting took me about 30 minutes

 

5) I washed them by adding 10ml of Opti-MEM and centrifugating at 300g for 3minutes

 

6) I discarded the supernatant and I re-suspended the cells (pellet) with 2.5ml of Opti-MEM and I incubated on ice for about 30 minutes along side with the electroporation cuvettes and my HCV in-vitro transcribed RNAs

 

7) I shared 500µl (0.5ml) of the previous cell suspension in 5 different tubes (for 5 reactions of electroporation) and I added 10µg (4µl) of RNA in each of them, I gently mixed it and put them inside the cuvette

 

8) I finally used the Bio-Rad gene pulser (under these conditions Voltage: 270V; Capacitance: 950 µF; Resistance: 100Ω; cuvette: 4mm) to get the RNAs inside the cells

 

9) The electroporated cells (all the cuvette's content) was put in a 10cm dish already having 10ml of complete medium (GIBCO) and 10µl of G418

 

 

My observations:

 

1) 24hours after the experiment, I changed the medium ; they were almost no cell present in all the 5 petri dishes

 

2) 48 hours post-electroporation (today), I do the checking and 4 of the 5dishes are at about 10/100 confluent and there is absolutely no cell in one dish (I mistakently pressed the gene pulser twice for this cuvette while conducting the experiment (I think it has broken the cell membranes))

 

 

I would be very happy if someone can confirm whether these observations are normal... 

Advises on how to improve the experiment are highy welcome

 

Thank you

Steve






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