How to remove the background and unspecific bands - (Aug/03/2011 )

Dear all,

Recently I performed the PCR, my target pcr product is 700bp, but always has another band is around 500bp.
I tried to do gradient PCR (annealing tempe: 40, 44, 49, 54, 58 and 60 degree), but the result is similar like before (shown graph in attachment).
The PCR programme is as follows:
1x 2 min 94 °C
10x 30 s 94 °C
30 s 65 -> 55°C
21 s 72 °C (30s per kb)
20x 30 s 94 °C
30 s (40, 44, 48.8, 53.6, 58.2, 60.1°C)
21 s 72°C
1x 3 min 72°C
The polymerase used is Verbatim from Thermo scitific. The gene used is codon optimized gene.

So how can I remove the background and unspecific bands like 500p? Do I add some DMSO for optimized gene?

Many thanks in advance!

Biogareth

Hi Biogareth

I am not to sure about your gradient PCR, however, if it is non specific products which are hindering your work, i have found that adding 8% DMSO (4 microL DMSO per 50 microL reaction) to the master mix helps no end.

Often before addition of DMSO i cannot get desired bands at the correct size, and afterwards no problem at all.

Agree with Chris22, DMSO will do the trick. But I use 5% in my reaction.

Am I reading your PCR protocol correctly and you do a touchdown for 10 cycles followed by your gradient? If so, my suggestion is to remove the touchdown part of your protocol and see how that goes.

1x 2 min 94 °C
10x 30 s 94 °C
30 s 65 -> 55°C is this part a touchdown?
21 s 72 °C (30s per kb)
20x 30 s 94 °C
30 s (40, 44, 48.8, 53.6, 58.2, 60.1°C)
21 s 72°C
1x 3 min 72°C

Adrian K on Wed Aug 3 16:34:55 2011 said:

I added DMSO inside, but the result didn't improve clearly.

leelee on Thu Aug 4 03:36:14 2011 said:

Thanks!
You are right, I combine touchdown with gradient PCR.
Following your suggestion, I delete touchdown part:
1x 2 min 94 °C
25x 30 s 94 °C
30 s 55 °C
21 s 72°C
1x 3 min 72°C
Here I used 55degree as annealing temp because it is in the optimal range from the previous PCR result.
I perform this PCR by two reactions (one with DMSO, one without DMSO), but the small band is still present.

I'd like to see a gel with just your template. Your gel shows bands > 2Kb, which are unlikely to be the product of a 21 second extension. I'm guessing you are using far too much template, and that the bands you see come from the template DNA, not your pcr reaction.

I had just missed out that you are actually using verbatim polymerase, which is a very high fidelity and fast polymerase with proof reading ability. If I guess correctly, you are using a thermalcycler with low ramp rate (~2C per second heating, ~1C per second for cooling). This is because your ~2kb band appears on the higher temperature rather than the lower one in your first gel, and since you using a low ramp rate machine, the annealing time and the extension time for higher temperature will be longer, due to the ramping which gives ~30 seconds longer for higher temperature gradient to maintain in place (i can elaborate further if my explanation sounds greek to you due to my bad expression).

Based on my experience using KOD polymerase (similar polymerase like yours), Such fast and high fidelity proof reading polymerase always gives me a hard time in optimization, really. This is because it doesn't really behave like normal Taq polymerase. Even under same annealing condition, Taq polymerase gives me my desired band but not in such high fidelity enzymes, and I use such enzymes only when I try to clone a large fragment (more than 2kb) and I don't want to wait for long time, else I will just stick to normal Taq. I have to reduce my extension temperature to 68C for optimal proof reading, and if I'm using a slow ramping rate machine, I need to reduce my extension time up to 5-10 seconds, to reduce the larger artifacts, and rising the annealing temperature up to 63C. Since your fragment is just 700bp, use a normal taq polymerase with Pfu blend will do.

Also, based on your label in your last gel, you are comparing the "GC-buffer" and "non-GC buffer" result, instead of "with/without DMSO", correct me if I'm wrong. And, according to the product insert http://www.sorvall.com/eThermo/CMA/PDFs/Product/productPDF_52581.pdf
the initial denaturation is 95C and denaturation is 98C. Is there any reason for you using 94C?

Biogareth,
Maybe you just cut and purify gel (700bp your desired size) and do pcr again
and then should be no more secondary band if you cut it properly...

Good luck

There's always a way to solve and many ways to stuck
we look on a way how to solve and we deal with many stuck
that's how and what research mean..so all i can say, good luck

Evanescence

Evanescence on Fri Aug 5 04:38:44 2011 said:

I tried to PCR again with purified target size, but still have two bands like before.