pGL-3 vector transformation - (Nov/17/2008 )
Hi, I am having problems with the transformation of DH5-a competent cells with ligated pGL-3 luciferase vector. I have been transforming them and spreading them onto amp+ agar plates. They just don't seem to grow. I dont know if it's whether the problems with the competent cells (I make them), or is it theres something wrong with the ligation reaction with the PGL-3 and promoter clone. Is there a way to see which is the problem and how to make the transformation process to be more efficient? Thank you
The way is to have controls for every step.
That includes whether your ligation is efficient, whether your competent bacteria, and transformation techniques are efficient.
You can make
1. Ultra-competent cells or
2. Electrocompetent cells,
but if there is a flaw in some basic steps, you will never get there. So, try to control and standardize all steps, and then go to more competent cells.
Try spreading you thawed competent cells on plain agar without amp. See if they grow. Then you can rule out if it's your cell viability problem. If not, make a new batch like cellcounter said.
If they do grow, try getting some actin/housekeeping gene to ligate as your +ve control and transform again. Technically it shouldn't be ligation problem as even though the insert doesn't ligate, empty-plasmid cells can still grow.
Best of luck
Hi, thanks for your reply. I have repeated my experiment yesterday and got the results today. I used the same ligated vectors from before, and I have a control sample, which is with PGL-3 vectors that hasnt been cleaved or anything. It came out that I have colonies for the control PGL3 vector sample, but not in my ligated vectors samples. That means theres prolly something wrong with my ligation PGL3 vectors, or my insert. The insert region for my vector is only 500 something bp. Which is not too large, any precaution to take when doing ligation? or what ratios of ligase buffer, vector, insert, ligase do you guys use and how do you incubate them??? Thank you
hi m also trying to do same ligation and my vector is also 500+ bp but i m not getting colonies i hvae tried several 100 of time but not working. any one have idea about this one.
i'm not familiar with this pGL-3, i do TA cloning. but the basic concepts are the same, i guess.
the fact that the e coli with uncut plasmid can grow means your cells and the plasmid are working fine. i think the problem is your ligation/preparation of your plasmid/insert. the cells can't grow probably because they don't have ampicillin resistance. the REs that you use, do they cut specifically where you want them? make sure they don't cut the ampR gene.
perhaps its better to call the promega tech support?