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Hello everyone, I am making a lentiviral vector that expresses 1 gene of interests by CMV promoter and 1 GFP marker by hPGK promoter. I want to know will this kind of combination work? They are both pol II recruiting promoters so it is supposed to see competition between these two, right? But we have numerous genes expressed in our body at the same time, how is the competition of pol II in there? Is it a problem of distance? Usually two genes will be much more apart in our chromosome than in the vector, right?
In my case, if I do this kind of cloning, when the sequence is getting integrated, how is the expression like? I guess I will have two transcripts, is this correct? No more others? Where would CMV-induced transcription stop? at the end of GFP or only the first gene? It depends on the location of poly A signal, right? what about if I use IRES for GFP? I read it somewhere that IRES is only working for translation from middle of an mRNA. If this is true, if there is a poly A in the end of my gene to stop IRES-GFP transcription, the GFP expression will not exist anymore, right?
Sorry for so many questions. Hope some one can be patient and answer me. Thank you very much.
#146809 a promoter question, two promoter cassetts-related expression
Posted
on 16 December 2012 - 11:12 PM
#107313 Problem in harvesting with MTT
Posted
on 19 April 2011 - 11:12 PM
gyma, on 15 April 2011 - 09:38 AM, said:
enthusiastic, on 14 April 2011 - 01:41 AM, said:
Hi all,
I am facing problem in harvesting the assay with MTT (5mg/ml).
My protocol is:-
Inoculate 100ul of cells at a density of 5 X 105 cells/ml.
Incubate for 24 hrs for attachment.
Discard media and then add 100ul compound with diff conc.
Again incubate for 24 hrs.
Then remove compound and add 10ul MTT and incubate for 4 hrs.
Centrifuge at 125g for 10 mins.
Remove MTT and add 100ul of DMSO to dissolve formazon crystal.
I am facing problem while removing MTT some of the formazon also come out in pipette n m not getting proper result. Plz help if you hv any other method of harvesting.
Thanx.
I am facing problem in harvesting the assay with MTT (5mg/ml).
My protocol is:-
Inoculate 100ul of cells at a density of 5 X 105 cells/ml.
Incubate for 24 hrs for attachment.
Discard media and then add 100ul compound with diff conc.
Again incubate for 24 hrs.
Then remove compound and add 10ul MTT and incubate for 4 hrs.
Centrifuge at 125g for 10 mins.
Remove MTT and add 100ul of DMSO to dissolve formazon crystal.
I am facing problem while removing MTT some of the formazon also come out in pipette n m not getting proper result. Plz help if you hv any other method of harvesting.
Thanx.
BTW, I never heard a method to remove MTT. Good luck
sorry I made a mistake. I didnot use DMSO to dissolve formazan, instead I used MTT lysis buffer composed of 2-propanol(500ml), triton-x(20ml) and 36%HCL(2ml). So I dont need to remove MTT or medium.
good luck.
