The DMSO affects the stability of the hydrogen bonds which are present between the two strands of your template AND between your primer and the template. Increasing the DMSO concentration has a similar effect on thermodynamics as increasing the annealing temperature. Its hard to pinpoint the exact reason of why you are getting noisy sequencing results (although 10% is a huge amount of DMSO). I would guess that you may have mispriming, so priming in unwanted area, so when you sequence this less abundant  and non specific PCR product (along with your wanted product) it generates the noise you observe. 

As for your exact question, what is the amount of DMSO you should use, I would say as little as possible!

DMSO will have a small effect at low concentration but why is it in the sequencing mix. Most purifications of pcr product will remove it. EXO-SAP will not remove it but 10% DMSO might inactivate the enzymes which might leave some of each primer present and this might produce 2 sequences. What is your purification method. Above 10% dmso inactivates enzymes even in pcr so generally I prefer less than 5% but if 10% works that is fine but increasing the annealing temperature and decreasing the dmso might be a good idea. If you attach the .abi sequence file it contains a lot of information that would make answering this question more accurate. I suspect that the amount of dmso left in the sequencing reaction is not the cause of the noisy peaks but more information is needed such as amount of dna, amount of primer and annealing temperature of the sequencing primer but ideally the .abi sequence file

• 10% DMSO is very high. The problem with DMSO at that concentration is that in conjunction with denaturation temp above 96C it can actually start to denature your amplitaq as well as your target template. If that are the case this would result in lower signal to noise and therefore noisier DNA sequencing ladder as you describe 
• in the past when I performed a lot of Sanger sequencing I would routinely add 5% DMSO
• If course with GC content greater than 70% you might not be melting all of your template and that could also explain your poor signal to noise 
• In the past when faced with such template specific issues I would 

There are reports that additives like glycerol and BSA spiked into your sequencing reaction can reduce denaturation of your Taq and thus offset those deleterious affects of high annealing temp and DMSO at 5% or more for a prolonged reaction. However I have seen no personal evidence for these claims 

finally for GC rich tempkates sometimes high GC content primers culminating in Tm values of 65-70C allow you to perform 2 step reactions at 65C where you anneal and extend in the same cycle.in the past  I have found this can help reduce noise from GC enriched templates 

Dear Laurence Sir,

While optimization my designed primers in Gradient PCR with standard human template DNA, I didn't use DMSO, due to low concentration in my subject sample, I used DMSO with what I standardized the annealing temp(61oC).

without DMSO. PCR product was gotten good intensity band. but the sequencing results got noisy peaks.

If I use 5% DMSO, sequencing results will be good and shall I increase annealing temp. also Sir?

I am having another query.

When I optimized other primers without DMSO by using standard human template 50ng DNA, those were good. but now problem with subject samples (low concentration) using 100ng DNA, added 10% DMSO in pcr master mix and annealing temp. when I optimized without adding DMSO, gotten good intensity bands along with non specific bands.

Please suggest for these and tell, what should I do?

Please do the needful.

Thanks in advance.

I will get back to you tomorrow with details 

Dear Yed

Apologies for late response

1. In over all summary I would use 5% DMSO (not 10%); make sure your annealing temp is 96C; not 94C or 98C; add an extra 5 cycles to both your PCr reaction and subsequent cycle sequencing reaction, i.e. if you currently use 25 cycles try 30 cycles (say for cycle sequencing); if you use 30 cycles for PCR try x35

2. For both your PCR reaction and cycle sequencing reaction before you do your main 3 step PCR or cycle sequencing (with extra 5 cycles) try an initial step of 96C for 3-5 min

Dear Sir,

Please find the attached file it has a table what are my primers and their details along with product length, few are previously optimized with human template standard 50ng DNA. Last 7 and 8 primers are optimized not worked yet and 9th one is getting non specific bands. I checked these primers BLAST also everything is fine.

After optimization, I did the polymerization of my subject (Patients DNA) with that annealing temp (which shows in table). not working. While this PCR, I kept one control which is standard DNA it was amplified remaining not working. I observed in gel it might be smear/no product/empty.

So once again I am going to be standardized these primers with different temp. in gradient PCR (applied Biosystem 96 well).

I could request you to give your valuable suggestion for these (which temp can I keep), and shall I use DMSO (for subject samples) for optimization.

Thanks in advance.

Talk soon 

Dear Yed

I notice that for a number of primers not working your Tm mismatches your annealing temp and products are > 3kb

To that end take a look at documents supplied and get back to me with any further questions

I will however say this:

1. For products > 3kb secondary structure is inevitable. Thus definitely include 5%| DMSO in your PCR mixture

2. The main thing however you need to do is switch to a highly processive polymerase (instead of Taq) to ensure that your large products are made in high copy numbers, i.e. that your PCR is efficient. To that end NEB phusion polymerase (also sold by a number of distributors) is your best bet:

https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase

3. Commenting in general, make sure your annealing temp= Tm of your actual primers to Tm-2C. Thus if you r Tm is for example 63C then your annealing would be 63C to 61C

4. Also ensure the Tm of your primers is preferable the same or no different than TM +/- 2C

5. To make sure your primers are thermodynamically matched and your PCR conditions are also accordingly matched use one of the sites for computing Tm specified in my extended SOP

http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/

6. For target regions with > 60-70% GC, especially if long you could try adding 1M betaine (final concentration) instead of 5% DMSO: See attached paper explaining why

7. Finally regardless of GC content sometimes something called touch down PCR instead of conventional 3 step PCR can sometimes yield sufficient amounts of PCR product when standard PCR fails or produces specific product but in low quantities; even when using a highly processive polymerase like phusion. To that end see attached

To that end see attached and also link below

http://www.nature.com/nprot/journal/v3/n9/full/nprot.2008.133.html

Good luck !!

Dear Laurence Sir,

I would like you to say thanking you for giving good suggestion.

I will try my level best.

If I have any quires, I will get back to you Sir,

Thanks.