If possible, I would like some explanation about luciferase assay
I'm trying to have several different portions of a specific gene promoter cloned into separate luciferase reporter vector pGL3-basic.
But I'm trying to request a service for this.
so I'll probably get 5 ug of vector-plasmid construct from the company.
Will this be enough for transfection into cancer cells and measurement?
If not, what could be a solution?
can the construct be amplified in bacterial culture?
I appreciate all the insights!
Hi Dave,
for your question: yes, you got just a little of plasmid (5 ug) so you can transform bacterial cells, and amplify your plasmid.
5ug is the amount that you will use for 1/2 transfection (depending on your condition) so what you need to do is:
1-get some competent bacteria and transform it with 50-100 ng. After overnight growth on the plate (with Amp as a selection Ab) pick 1 colony and put in 3ml overnight LB
2-from the 3 ml culture, you can use 1ml to make glycerol stock to store at -80
3-from the remaing , you make a 50 ml culture to grow overnight
4-from the 50 ml culture you extract your plasmid (midiprep from Qiagen or similar)
All the methods you can easily find online, you'll need to buy only the midiprep kit (if you want to save money)
hope this helps
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