Non-specific bands... what do you think is the reason in this case?

[Azifri]

Hello! I am new in this forum so hope its the correct place to post this.

I wanted to share with you the picture of an agarose gel I made yesterday and know your opinions about the non-specific bands I see here. The story is: I made a PCR to amplify certain part of a plasmid. I used iProof high fidelity DNA polymerase, and as it was my second time using it I did all the reactions in triplicate to test 3 different amounts of magnesium.
The primers used have a "tail" that may form hairpins, but this was unavoidable as I need to attach a certain sequence to the PCR product.

Size of the plasmid: 6.2 kb
Size of the expected product: 1.5 kb

Gel: 1% agarose in 1X TBE buffer.

Picture (remove the @@@ before opening. Sorry, the forum was not allowing me to post links or images): ht@@@tp://img36.imageshack.us/img36/...el02012012.jpg

Thanks for answering! If you need more info let me know

[Azifri]

Originally Posted by Azifri

Hello! I am new in this forum so hope its the correct place to post this.

I wanted to share with you the picture of an agarose gel I made yesterday and know your opinions about the non-specific bands I see here. The story is: I made a PCR to amplify certain part of a plasmid. I used iProof high fidelity DNA polymerase, and as it was my second time using it I did all the reactions in triplicate to test 3 different amounts of magnesium.
The primers used have a "tail" that may form hairpins, but this was unavoidable as I need to attach a certain sequence to the PCR product.

Size of the plasmid: 6.2 kb
Size of the expected product: 1.5 kb

Gel: 1% agarose in 1X TBE buffer.

Picture (remove the @@@ before opening. Sorry, the forum was not allowing me to post links or images): ht@@@tp://img36.imageshack.us/img36/...el02012012.jpg

Thanks for answering! If you need more info let me know

For the image:

A: complete reaction
B: control without DNA
C: control without polymerase
D: control without DNA and polymerase

[admin]

Hi Azifri,
welcome!
for non-specific bands its best to start from the top and go down the list.

Do you have your protocol? How much DNA template did you use? What is the GC % of your region to amplify?

If it is over 60% GC rich then you can try DMSO 1-5% in your reaction it helped me with such tough sequences. For self-annealing primers you can try higher temperatures for annealing to minimize self-annealing.

Some tips for non-specific amplification

• Use less DNA template (your plasmid seems to show up but may not be an issue depending on what you do next with the reaction)
• Use less primer (not sure what your final conc. was of primer)
• Decrease the annealing time / increase annealing temperature
• Use touchdown PCR
• Decrease the extension temperature to 62-68ºC
• For high GC regions use DMSO 1-5% (final conc)
• Increase the KCl (buffer) concentration to 1.2x-2x, but keep MgCl2 concentration at 1.5-2mM
• If none of the above works: check the primer for repetitive sequences (BLAST align the sequence with the databases and use primer software to check self-annealing) and change the primer(s)
• Use a combination of some/all of the above

your lane C: control without polymerase looks like it has a lot DNA in there, your plasmid 6kb seems to be showing up along with its nicked or partially ss plasmid up higher possibly. These may explain your extra bands.

[Azifri]

Hello Admin! Thanks very much for the quick answer and the tips, I certainly will use some of them.

Ok, the protocol was:

Initial denaturation: 98ºC for 30 sec
Denaturation: 98ºC for 30 sec
Annealing:60ºC for 20 sec
Extension: 72ºC for 22 sec
(30 cycles for these last 3 steps)
Final extension: 72ºC for 10 minutes

I used 10 ng of DNA in a 50 ul reaction, as suggested in the manual, but I agree with you that maybe it was too much, according to what is seen in the gel.

The GC content of the region to amplify is 55%, I think I will try DMSO, and looking around in the lab I found a "GC buffer" for PCR (seems that the exact composition is a mystery from the company). Maybe I will give it a try.

Primer concentration was 0.5 uM, do you think using less may improve the reaction?

I also thought that the upper two bands are maybe from the plasmid.
On the other hand, the band between 0.6 and 0.8 kb confuses me a bit. First I thought that it may be primers, but if that is the case there should be the same in the control too (as primers were added in all the cases). Maybe its some non specific amplification?