Amplify fragment in plasmid? - (Dec/08/2011 )

Hi,

I have several times tried to amplify a fragment in a plasmid. Every time I getting smear and unspecific binding. I have try to change salt, add DSMO, different concentrations of template.

My supervisior gave me this template, which is a plasmid (vector) that contains an insert in size 640 bp, the vector is about 3000 bp. I really like to solve this problem but I dont know how.

Can some one give me a hint of what to do, please.

Hi,

Have you tried changing the annealing temperature?Sometimes you can get rid of nonspecific binding by taking two temperatures for annealing. In this first 2-3 cycles are given on different annealing temperature and then rest of the cycles are performed on the specific annealing temperature. you can give it a try, if it works.

amj2

Hi and thanks for answer.

Yes, I have tried annealing temp: 56, 60, 65 and 70 degrees. Tm for primers are 80 and 75 degrees. Everytime and whatever I try still same result. And everytime that bend from the plasmide appear. I basic question if it is more difficult to amplifie a single segment when it is in a vector? Would it be easier if the template just contain the fragment of interest. My supervisior gave me one tube 3*X (X=gene of interest) in pGreen and told me to amplyfie and purify X.

Some questions that come to mind:
Are you sure your primers are good? you can always redesign and try different primers.
Are you trying to amplify the whole gene or just a part? You might need to take into account where the insert binds the vector (and to each other, is it a concatemer of your gene x3?)
Are you sure is the right template? (if having problems consistently it might be worth sequencing the vector to be 100% sure)

I think the more detail you can provide us, the best we will be able to help.

Alternatively, if you think that amplifying from the vector is an issue (and more because of the 3X issue), you could try digesting the insert out and then use that as template for PCR.

You may be using too much template. Try diluting the template by 10x and 100x. Do you have sequence for the plasmid? How certain are you that the plasmid is really what you think it is?

I would also try a lower annealing temperature, perhaps 52 and 50. The Tm of your primers seems suspiciously high. Are you including a 5' extension in your Tm calculation? If so, your annealing temperature will have to be much lower.

I would reiterate the suggestion to redesign primers. When there are difficulties you can't otherwise explain, this often works.