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Re: Trouble with Lentivirus transfection
Posted by: krisha (IP Hidden, Unregistered user, )
Date: June 15, 2005 02:26PM
Thanks for the suggestion...I did try with another compound called Polybrene (6ug/ml) previuosly.
Let me try with DEAE-dextran next time. thanks again
Re: trouble with siRNA transfection
Posted by: thomas (IP Hidden, New member, 5)
Date: July 3, 2005 02:38PM
I don't know about Genecarrier-1 or Genecarrier-2, but we have used RNAiCarrier from epochbiolabs. My lab has tried numerous times to transfect 4 different cell lines under the conditions of their protocol and have tried to optimize it for our use. We have increased and decreased the amount of reagent and we consistently only get a 15-30% transfection efficiency in the FAM siRNA transfected cells vs. the control cells. We know this FAM siRNA works great with our FACS machine using HeLa cells with Lipofectamine 2000 transfection reagent - we get a 90-95% increase in fluorescence.
Re: trouble with siRNA transfection
Posted by: msp (IP Hidden, New member, 2)
Date: July 15, 2005 09:12AM
Hi,
I'm thinking of transiently transfecting mouse pre-B cells with siRNA. Has anyone tried it? If yes, which transfection technique would you recommend? Many thanks!!! Edited 1 times. Last edit at 07/15/05 09:13AM by msp.
Re: trouble with siRNA transfection
Posted by: dr Melton (IP Hidden, Unregistered user, )
Date: July 15, 2005 01:04PM
We did siRNA transfection of 1881 mouse pre-B cell line with RNAicarrier and got about >80% gene knockdown (~60% efficiency using FITC-siRNA) based on RT-PCR and Western.
However, in the case of shRNA expression plasmid transfection on this cell line, no liposome reagent work for us, so you need to do electroporation. good luck.
Re: trouble with siRNA transfection
Posted by: msp (IP Hidden, New member, 2)
Date: July 15, 2005 03:32PM
Thanks for your reply!
Re: trouble with siRNA transfection
Posted by: albertotom (IP Hidden, New member, 1)
Date: October 14, 2005 03:47AM
dear Colleague
we are attempting to transfect jurkat with GFP-neo vectors unfortunately we didn't find detailed protocols for transfection and selection of stable transfectants we tryed jetpei and superfect with good results on fibroblast but without success in jurkat we are now evaluationg elettroporation, but probably we still have poor results (we performed selection in 96 wells plates with 10000 cell/well and with 1 to 2 mg/mL og G418). After 10 days, there are still many viable cells but none of them seem to express GFP at higher extent as non transfected controls. Can you please send me a detaile step by step protocol. I see you worked best with gencarrier 2. thank you for any help yours sincerely A. Tommasini
Re: trouble with siRNA transfection
Posted by: hutman (IP Hidden, New member, 5)
Date: February 18, 2006 10:01AM
Hi,
I have excellent experience with siRNA and shRNA knock-down electroporation using GTporator solution. It worked for almost every cell type I have tried with more than 80% efficiency. I is quite cheap and they provide a free sample for testing. See yourself here [www.gentrend.cz] Hutman
Re: trouble with siRNA transfection
Posted by: anivdas (IP Hidden, New member, 1)
Date: February 22, 2006 12:03AM
Hi all,
I am having weird problems with siRNA. I am using a siRNA oligos (from Dharmacon). I tried to deliver siRNA using electroporation method. We have been getting good transfecdtion efficiency with GFP vector. Recently I have been experiencing an increased expression of the gene which I am trying to knock down. I used to get atleast 70% knockdown with same siRNA before. Why do the gene expression (by RT-PCR analysis) increase instead of decreasing? I am really frustrated with this problem? Can anybody help me? Thank you very much adas
Re: trouble with siRNA transfection
Posted by: dkhurtado (IP Hidden, New member, 5)
Date: June 7, 2006 02:54PM
Who makes GeneCarrier-1? I googled it to no avail....
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