I would use 5% DMSO for GC rich templates

Farhan, few questions:

1. what is the length of your product?

2. how GC rich is your sequence, ie ~TM for your product?

3. how far out from your GC-rich region are your primers?

Amit

I would use 5% DMSO for GC rich templates

I work with 70% GC-rich organism. Our primers are typically in the range of 72C annealing temp for 20-24 bp. We use in-house magic enhancer made of betaine (Sigma), DMSO, DTT and BSA. It works great. Try doing a titration of magic enhancer in your PCR to see what concentration works for you and from there proceed onwards.

I cloned a 74% GC PCR product that was around 840 bases. A combination of DMSO and Betaine will work. Again, titration would be helpful to get a clean product with no smears. Best of luck. You can do it..

I am using the OneTaq hotstart PCR from NEB in a regular PCR w good Luck. I use the 5x buffer and the 10%Enhancer.

cat # M0480L 1000rxns but they will send a sample to test first and you can buy smaller kits.

Dear Farhan

u can try these tings:

1. denature template at 96 degrees for 3 minutes then add pcr mix over the template on pcr machine.

2. add 7 cycles of annealing at 70 degrees according to the size of product..........its important.

3. use DMSO and formamide 5% and 1.25% respectively.

4. the next cycle will be the same for mix having just DMSO as u do routinely and if u trying along with formamide, annealing temperature wil be decreased 20 degrees.

here is the programme:

1. 96_______5 ( 3 min for templ denaturation, 2 with mix)

2. 96_______45sec

3. 70_______20sec

4. 72_______according to size of product

go to step 2 n repeat 7 times

5. 96_______45sec

6. 5 degrees less than the manual annealing temp with DMSO n 20 degrees less if u using formamide____20sec

7. 72_______according to size of product

go to step 5 n repeat 33 times.

8. 72_______10minutes (final extension)

i tried this protol to amplify 83% GC rich product.

Beat of luck!!!!

Like the others persons before me told you, I advise you to use DMSO or betaine which is better. Use different concentration of DMSO or betaine and use the smallest one. Indeed, DMSO could lead to polymerase errors.

Increase denaturation step to 98°C and use a nested PCR.

I would highly recommend Epicentre MasterAMP. It is a well known kit to treat similar problems. You can as well try the FAILSAFE Trial kit to find what can be the best combination to setup your PCR reaction.

This may have the exact amount of Betain and DMSO as per your reaction.

With regards,

Gaurab

Http://www.epibio.com/item.asp?id=294

Dear Farhan, first you change the PCR condition specially Amplification Temperature and increase the Cycle No. If you again suffer with same problem then you change PCR mix composition and try . Because some time amplification occurs after 30 cycle, some time 7 -10 degree below and high Tm than actual Tm. I hope you will get the result.

when I worked with GC rich template DNA (such as Mycobacteria ), I found useful the use DMSO (3% to 5% max ) added in my PCR mix . Then I found out the product :"GC-Melt Reagent" by Clontech, the results were better than those obtained with DMSO. you can try it !!

You can try with 5 - 10% glycerol and higher temperature of annealing. I would suggest you to combine this with use of RedTaq (WVR - Denmark). This mastermix amplify really strong and you need good optimization of reaction conditions.

Good luck!

Dear friend you first you confirm function of DMSO in PCR. after that you add DMSO , because DMSO will make mutation in pcr mix , so that wrong base pair will our

gc rich pcr system from Roche, as is

HotStart PCR, DMSO up to 10% (ie.:APOE polymorphisms). In literature: Hube et al. Mol Biotech 2005;31:81-4.; Musso et al. J Mol Diagn 2006;8:544-50.

Farhan, it is best you try any of the useful suggestions given above and share your success or otherwise so we know what more to suggest next.

Good luck.

Apart of all what colleagues said did you check for your primers

I think using the magic buffer supplied with KOMABIOTECH PCR master mix will be helpfull to you.

http://www.komabiotech.com/product/product_detail.php?item=K0567510

Hi Farhan,

I'm working with rich CG template and initially used a specific polymerase for this sequences.....never worked!!! Then I tried to amplify using regular HotStart and betain 2M....perfect! I'm using this material for sequencing and works pretty well.

Sorry. My grandson was helping me send too soon. In a sample 800-1000 bp long I did not change the chemistry of the protocol. No DMSO additions etc. The key for me was raise the annealing temps in a TD program. The temp above (72 deg) seems plenty high. The viruses I worked with usually reponded quite well to 66 or 68. You could run graduations to find the right annealing temp.

Thanks! Great suggestions. I tried KAPA3G GC rich Kit with 5% DMSO and it worked for a few of my templates. I also tried the slow-down method in which heating and cooling ramp rates are reduced by half with out dc7GTP, but that did not work and I did not bother repeating.

Thanks Toni, Terry, Sabine, Aaron, Yahia, Farid, Nicholas, Ferenc, Doinita, Rama, Amara, Mukesh, Sugata, Ahmed, Rebecca , Graham and Amit.

Hi!

I see that there are a lot of suggestions about usage of well known additives like DMSO, betaine or special kits designed for amplification GC-rich DNA templates. However, in the recent paper by Zhang Z. et al. "Enhanced amplification of GC-rich DNA with two organic reagents" BioTechniques 47:775-779 (September 2009) the authors amplified 104 GC-rich human genome amplicons and showed that in some cases ethylene glycol and 1,2-propanediol are more effective than betaine. Have a look at this paper, who knows may they have managed to amplify one of your hard amplicons.

In my experience, GC-rich templates can be amplified by decreasing total Mg2+ concentration to 0.5 mM. I think this is due to destabilization stable secondary structures. May be combination of decreased Mg2+concentration with one of the additives like betaine will help.

Also I suggest to check integrity of primers by polyacrilamide gel electrophoresis. If you'll see nothing or a smear - the matter is in the primers, not in the PCR conditions. Some colleagues in my lab encountered with that fact. In one case there was no an oligo at all.

Moreover, you can check if the site where your oligos should anneal have strong secondary structure or several closely situated structures. Such an analysis you can perform here http://mfold.rna.albany.edu/?q=mfold/DNA-Folding-Form. If you'll found that one of your primer annealing site contains strong structures and you have a choice to move its position you'd better do it because at conditions that can disrupt strong secondary structures the primer is unable to anneal to the site.

Good luck!

Very practical suggestions Dmitry. Thanks!

Hallow to every one in this helpful supporting group, i do appreciate their help with valuable fruitfull informations dealing with their works. iam just bigginer in this field and of course i got alot of knowledges from all comments and i learned from that. Thanks to all.

I guess the problem is solved and that's great. For the sake of completeness of this discussion, I would also suggest PCRX (x is in suscript format) Enhancer System from Invitrogen (Catalog Number: 11495-017), which I have used successfully in my published research.

Thanks Tomasz!

i'm working with CG rich template (66.6%)and i amplified 2.3kb. by the additon of 5% glycero to the reaction with lowering the annealing. If you used DMSO or betaine you must add additional volume of polymerase because of these additive inhibt 50% of the enzyme activity. Finally i advice you to used absolute glycerol

Hi,

I would like to suggest something completely different. Resuspend desicated GC rich DNA templates in ultra-pure sterile water.. In completely deionised sterile water the DNA the double stranded native form of the molecule will be less stable than in an ionised buffer.

Place a 20 microlitre drop of this solution onto a flame sterilised cold microscope slide. Holding a slide with a sterile forceps waft it through a blue bunsen flame for one second at a time inside a laminar air flow work station.. Allow the fluid drop to dry completely on the now hot microscope slide.

Now place the slide onto a sterile cold metal plate of foil plate to cool inside the laminar air flow work station. When cooled add 20 of deionised ultra-pure water to resolubilise the denatured nucleic acid. This template material will now be quite easy to amplify nucleic acid fragments from as the flame treatment will fragment and permanently denature the large genome into elements more amenable to DNA amplification.

I think this simple approach will yeild some interesting results for you in your amplification of segments of GC rich genomes from eubacteria.

Please let me know how it works out for you.

Increase denaturation step at 98° for 10'' and try Takara TAQ.

Hai increase denaturation temperature at 98 -100 for 10- 15 minutes

i agree with Massimilliano chetta answer , In crease denaturation temperature

I think the role of ultra high fidelity polymerases such as Q5, Phusion, KOD Fx, Deep Vent cannot be ignored in high GC situations apart from the routine optimization by DMSO, Betaine or an increase in denaturation temperature...If KAPA2G failed you I guess Q5 would be a good choice.

Dear Farkhan, I recommend Roche not only because I represent Roche but because Roche really has premium reagents that seldom fail. Most of them are functionally tested meaning that they do work under specifications. I attach a publication which is explanatory for GC rich target approach in general, whether you can see that a quality reagent, among other conditions, can do the job. 

For the short templates are easy. But for the templates longer than 5kbp are very hard

what is your idea about amplification of GCC repeat in fragile X disease??