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How to remove the background and unspecific bands


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#1 Biogareth

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Posted 03 August 2011 - 07:34 AM

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 -> 55C
21 s 72 C (30s per kb)
20x 30 s 94 C
30 s (40, 44, 48.8, 53.6, 58.2, 60.1C)
21 s 72C
1x 3 min 72C
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

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#2 Chris22

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Posted 03 August 2011 - 07:55 AM

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.

#3 Adrian K

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Posted 03 August 2011 - 08:34 AM

Agree with Chris22, DMSO will do the trick. But I use 5% in my reaction.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#4 leelee

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Posted 03 August 2011 - 07:36 PM

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 -> 55C 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, 60.1C)
21 s 72C
1x 3 min 72C

#5 Biogareth

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Posted 04 August 2011 - 02:18 AM

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



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

#6 Biogareth

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Posted 04 August 2011 - 02:27 AM

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 -> 55C 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, 60.1C)
21 s 72C
1x 3 min 72C



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 72C
1x 3 min 72C
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.

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#7 phage434

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Posted 04 August 2011 - 04:59 AM

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.

#8 Adrian K

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Posted 04 August 2011 - 11:02 AM

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.c...ctPDF_52581.pdf
the initial denaturation is 95C and denaturation is 98C. Is there any reason for you using 94C?
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#9 Evanescence

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Posted 04 August 2011 - 08:38 PM

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

#10 Biogareth

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Posted 05 August 2011 - 01:15 AM

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



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

#11 Biogareth

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Posted 05 August 2011 - 02:02 AM

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.c...ctPDF_52581.pdf
the initial denaturation is 95C and denaturation is 98C. Is there any reason for you using 94C?



Thank you Adrian for explanation.
Recently our lab change to use Verbatim for HF PCR because of financial reason. You are right this enzyme probably is too fast to control PCR reaction, and resulting in unspecific bands. Because I will use a mutagenic primer afterwards, I wonder that Taq enzyme will influence the high fidelity or not.
As far as the annealing temp I used, this PCR programme works well with my collegues.

#12 Evanescence

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Posted 07 August 2011 - 02:14 AM

Biogareth,

How many cycles you used to run??
Try to make it less cycle....if u doing 30cycles, try make it 25...it can reduce noise :)
or try to extend the denaturation time (maybe it helps)

I dont like to suggest this, but maybe you also need to redesign primer to get more specific one

Regarding to pcr your purified gene, did u check the band (after you purifying your gel) before run pcr? i am afraid, some of 500bp band was cut and and got purified too...

#13 FMK

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Posted 01 October 2011 - 06:58 PM

I would gel extract both bands TA-clone them, sequence and then decide what is going on. May be everything is fine with your PCR but you are dealing with a heterozygous deletion... Or if the second band is completely off target you should just redesign one of the primers... Sequencing will make it all clear!

Edited by FMK, 01 October 2011 - 06:58 PM.





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