Thread: Transfection assay

Hi everyone!


I have to do some transfections assays, and I have a question regarding the empty vector should I use ... I will use a vector pGL3-luc (IL-2 promoter). So my question is, what empty vector should I use?, What does empty vector?
Please help me!

why 3 identical posts ? i've taken the liberty of deleting the other two

Originally Posted by kitty

Hi everyone!


I have to do some transfections assays, and I have a question regarding the empty vector should I use ... I will use a vector pGL3-luc (IL-2 promoter). So my question is, what empty vector should I use?, What does empty vector?
Please help me!

Your question is unclear.

Seems like you give the answer first, and then ask if we read the first part. "I use pGL3-luc, what do i use?"

An empty vector is a carrier of a vector that has not yet anything inserted inside. Usually without any elective properties, but with some selective properties, an origin, and as many cutting sites as needed. I suppose you want to transfect prokaryotes (E.coli) with a vector that is a plasmid?

Simply use a strain of e.coli that has a plasmid inside with a resistance factor. Then grow them on ampicillin (to multiply the plasmid). Then kill off the bacteria, and purify it, so only small circular dna is captured. Then cut the plasmid with restriction enzymes, paste your insert (actual vector, the luciferase) inside, and transfect into a similar E.coli strain that does not contain a plasmid. Use heatshock, or a specific lysosyme to get the plasmid inside the bacteria.

Voila..

Sorry my English is not perfect yet. Let me explain my question again:
I have to do some transfections assay (for a human cell line) in 6-well plate, I will co-transfected two plasmids: pGL3-IL2-luc and internal control pGL4.74 [hRluc / CT]
For the assay is necessary to transfect the same amount of DNA per well, and the literature recommended 1 ug of DNA transfected per well (in a 6-well plate). That's why it SHOULD transfected with another plasmid (plus pGL3-IL2-Luc and pGL4.74 hRluc / CT) for the final amount of DNA (to make 1 ug), and recommend to do an "empty vector" which I do not know I should I use for my case!.
I read that some laboratories use pCS2P +. But if the backbone of renilla and luciferase ( that Im using) are pGL3 and are pGL4, respectively. Is it more advisable to use a pGL3 vector without the luciferase coding sequence for completing the amount of DNA per well?

Honestly, i have no idea what to tell you.

Eiter it's way above what i know, or your explanation is not good enough for me to help you.

Transfecting human dna can only be done with viral vectors. Human viral vectors, and those are hazardous to humans (who knew), so i strongly disadvice you from using them. Secondly, i think you want to transfect the vectors themselves. Using an empty virus case, and transfecting your non-infective dna sequence in there. Although you should provide your strand parts where it can integrate with the nucleus.

Seriously though, i have no knowledge about this, i'm just guessing..