2 possible ways of generating a smear in pcr are

1 one primer only is working and the other creates a smear. Check that the primers in the smeared pcr have exact homology with the template dna.

2 There is too much template dna and the sample is over amplified leading to the pcr product self annealing and producing many different size products. Pcr works well on the number of copies of the template dna not the total amount of dna so as plasmid is quite a small template it is easy to add too much template. Then theoretically 10 cycles of pcr is 1000 time more product and 20 cycles is 10exp6 times more so it might be worth checking the relative amounts of dna in the 2 samples

Paul Rutland The concentration of the Vector DNA of the first sample is: 283,4 ng/μl, of the second: 748,7 ng/μl. I deluted 1 μl of the DNAs in 283,4 and 748,7 μl Water. For the 20 μl PCR assay I then took 0,4 μl. Is this too much DNA?

Hello Helia Tavabe Ghavami ,

using the formula below and assuming a vector size of8000 and double stranded dna

The DNA copy number calculator works by using the following equation.

📷 ng x 6.023 x 10exp23/length x 10exp9 x mass of dna

Further information for each component of the DNA copy number calculator is also listed below.

· Amount (ng) – The amount of DNA, in nanograms (ng), in the tube.

· 6.022 x 1023 – Avogadro’s constant. This represents the number of molecules per mole.

· Length (bp) – The length of DNA, in base pairs (bp), in the template.

· 1 x 109 – A factor used to convert to ng.

Mass of DNA bp – The average mass of a DNA bp is either 660 (dsDNA) or 330 (ssDNA) g/mole. This value depends on what is selected as the type of DNA in the calculator

both of your samples give a value of about 5x10exp10 copies which I would normally say is too much BUT your second sample is working at this level so what is the difference?

I wonder if actually some of your dna is actually total genomic dna so very large and complex possibly with viral dna motifs which are binding your primers and producing random sized amplimers as genomic viral sequences can often be inverted and may produce a pcr product. It would also mean that your vector concentration is very much lower than you think

If this is the case then we do not really know how much of your dna is plasmid so the best that I can suggest is either to run known amounts of vector1 against a known pure vector on agarose gel or run dilutions ( 1/10) from your concentrated stock in the pcr reaction and see if any dilution looks like amplifying well

Paul Rutland Thank you for the advice. I actually tested the Vector DNA by mixing 2 µl of the DNA with 4 µl water and 2,5 µl Loading Dye. The result was good. I saw a clear band at 12.000 bp. Also another time it functioned (was an exception), although it was a very small band. I will add a picture.

Also I use Phusion Polymerase with GC-Buffer. Thought i mention it, maybe it is related to the problem.

Thank you again for your advice.

Thank you for the picture Helia Tavabe Ghavami . If the first sample beside the ladder is your problem amplification then it looks like there is a strong primer dimer band. It may not look strong but small dna does not trap much EtBr so if you cab see it then there is quite a lot. It amplifies well so removes a lot of primer leading to poor proper amplification. You could try two amplifications with half and quarter the amounts of primer ( to try to stop early formation of PD ) and also increase the extension time to maximise the long amplification product even though I am sure that you are using the recommended extension time for this enzyme according to the manufacturer.

Phusion is a good enzyme and high GC buffer works well with both high GC and high AT sequences and it also works with your other amplification so I would trust it

Paul Rutland I use 0,4 µl for the 20 µl assay. I can try next time with 0,2 µl or 0,1 µl. I added a picture of my PCR programm. I changed the annealing temperature to 60 degree (not 62 degree anymore).

Thank you for your help.

Try 10 minutes extension as well, just to maximise the chance of the long product amplifying better Helia Tavabe Ghavami

good luck

Paul

Paul Rutland Hello, thank you a lot for your help. I just looked up the Agarose gel result, where I loaded 1 and 2 μl of my Vector DNAs and added water and Loading dye like I described before. I found that each of them has a faint band and a clear one. But I didn’t use primers, so they’re not primer dimers. I wanted to show the picture before I repeat the experiment again.

Thank you in advance.

Helia Tavabe Ghavami I think that the 2 large bands are just different conformations of the plasmid dna. You will only get primer dimer during pcr and it is less than 100bp in size. You clearly have large amounts of plasmid dna that is not contaminated with rna or bacterial genomic dna which is a very good thing. good luck

Paul

Years ago I used the Phusion enzyme to amplify a large (16kb) piece of the Adenovirus genome. I, too, had a problem with smears in the products. My smears were mostly high molecular weight. The recommended trouble-shooting is to reduce the concentration of enzyme, shorten the extension time and reduce the number of cycles.

My experience was that reducing the polymerase made the biggest difference. If memory serves, I was using 0.1units in 50ul reactions. Extension time made a smaller difference, but I don't recall my extension times back then. Currently, I am amplifying much smaller products (about 3-5kb) from plasmid and use 60 seconds with good results. The number of cycles did not make a huge difference in my hands, but it might help for you. The other thing to try is amplifying with different concentrations of DMSO added. I have had more success with optimizing the DMSO concentration than with the Mg++ concentration.

One other thought... are you using the Hot Start version of the polymerase? That can help to eliminate smears and background, and it is worth the small extra expense in my opinion.

Can you try to dilute the plasmid before amplifying, do a couple of tests diluting by half and 100 times? Often an excess of template DNA can give results like what you got!

Alternatively you can try using a working solution of 1.25 mM magnesium chloride because excessive amounts of Magnesium Chloride in PCR reactions lead to non-specific binding of primers.