It may be that the circular template forms a secondary structure that hides the primer binding site(s) so you get a low efficiency pcr that then amplifies a band as expected. The linear dna may amplify very much more efficiently so produces a lot of amplimer early in the cycles and then the ampunt of amplimer allows amplimer to amplimer annealing and results in a long smear of many different length concatamers. You may just be starting with too much template for an efficient pcr. How much template are you using and how many cycles? About 30,000 copies is a good amount to amplify but when using small molecules like plasmids it is very easy to start with too much dna

Hi Rutland,

Thank you for your input. I used a range of template (0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, and 10ng). I got smear from 10ng to 1ng template but nothing from 0.5ng to 0.01ng template (not even a smear). I am using 25 cycles of amplification. Do I further need to reduce any of them?

Normally I would say that you should be amplifying picogramme to femtogramme amounts of plasmid dna to get a reasonable number of copies to amplify but in this case you say that the smear vanishes at.5ng so I am wondering if the pcr is working at all. One single primer annealing and extending will also produce a smear of amplified material and I think that you need to test whether both primers are annealing and extending, Check the primer sequences and try an amplification at an annealing temperature 5c below the annealing temperature of the sequence specific bases of the lowest annealing primer on both linear and circular dna at 1ng and 1picogramme amounts. Until you have good reason to believe that the primers are both working well it is difficult to sort out the best amount of template to use

The amplicon is being made by using circular plasmid as a template. I am using the same conditions for primer annealing in case of linear template but failed. What do you think

Can I be clear about amplification? Are you getting any pcr bands of the right size with either circular or linear dna as template ?

Are the primers completely complementary to the plasmid sequence or do they have 5' additional sequences for re ligation and is the annealing temperature calculated onlyon the complementary sequence?

Are these new primers or old and inherited from other researchers and do you have a dna template that they do amplify as a control sample for the pcr reaction?

When you linearise the vector+insert is there any possibility that the restriction site exists either in the inserted sequence or anywhere else between the pcr primers which would cause a pcr to fail

Yes. I get specific PCR bands of right size with circular DNA as a template (but not with linear DNA as a template).

Primers do have additional sequences (for ligation) and annealing temperature was calculated based upon the full-length of primer (complimentary + additional).

These primers are designed by us (not sure about others). Yes, these primers can amplify control sample for PCR reaction (circular DNA as a template)

I am not linearizing the vector/insert by restriction digestion. It is a linearized DNA which resulted as a PCR product after using circular DNA as template.

Thank you Nasir so we know that the primers and pcr reaction works and the problem is with the template dna.

I would amplify 3 samples 1ul of 1/100, 1/1000 and 1/10000 dilution of the linearised dna and amplify in the presence of dmso 5% and betaine 1 molar final concentration, If your enzyme comes with a high GC buffer then just use this to open up the secondary structure of the linear dna, Drop the annealing temp by 5c for this amplification

I am surprised that the first pcr worked as the annealing temperature must have been very high and I worry that you have no amplification from the plasmifd and tou are just seeing a plasmid dna band from added template. I would also calculate the annealing temperature without added non complementary bases and run the first pcr again at the lower annealing temperature. You can check this thought by runnung a gel with diluted plasmid at the same amount as in the pcr as a control beside your amplified product from the plasmid pcr

Hi Paul,

1) I will run PCR with lower amount of linear template (as suggested by you), 5% DMSO, and high GC buffer.

2) I have done gradient PCR with a range of temperatures which also include the annealing temperature without adding non-complementary bases.

3) I have confirmed the successful amplification from plasmid template by changing one of the primers as I got bands of different sizes. I will further confirm as you mentioned.