Hi Isha,

there are different possibilities you can choose from or may have to combine with each other.

First I do have 3 questions though:

• is it a GC-rich DNA region eg promoter/CpG island you want to amplify?
• Do you need it to be 100% accurate eg you would want to use a proof reading taq?
• are there repetetive sequences in the DNA region you want to amplify?

And here are my suggestions:

• you may want to redesign the primers.
• I usually run a normal PCR program for amplifying DNA or cDNA fragments even if they are 1500 bp or so (for non-proof reading I usually use the Hot Star Taq from QIAGEN): 95°C 15'; (95°C 30'', 56°C 20'', 72°C 90'' (eg 1 min per kb, so I would use 60'' for your 858 bp fragment)) 40x; 72°C 10'; 4°C hold
• As proof reading taq I use the AccuPrime proof reading high fidelity taq from Invitrogen according to the manual
• Concerning the taqs etc. - I have made the experience that some fragments do not amplify with one taq but nicely amplify with another and vice versa so buying a different taq may work
• if you are amplifying GC-rich DNA you may want to add  5% DMSO (final concentration)

Ok, that's it right now.

Hope it's helpful.

Best and all the Best for 2016,

Christiane

PCR using gDNA as a template can be tricky. The reason for this is the sometimes poor quality of the gDNA preparation (presence of contaminants, etc.). A common error is that people use too much gDNA as template. Less is more when it comes to PCR with gDNA as a template. 

Cheers, Christian

Hi Isha,

there are different possibilities you can choose from or may have to combine with each other.

First I do have 3 questions though:

• is it a GC-rich DNA region eg promoter/CpG island you want to amplify?
• Do you need it to be 100% accurate eg you would want to use a proof reading taq?
• are there repetetive sequences in the DNA region you want to amplify?

And here are my suggestions:

• you may want to redesign the primers.
• I usually run a normal PCR program for amplifying DNA or cDNA fragments even if they are 1500 bp or so (for non-proof reading I usually use the Hot Star Taq from QIAGEN): 95°C 15'; (95°C 30'', 56°C 20'', 72°C 90'' (eg 1 min per kb, so I would use 60'' for your 858 bp fragment)) 40x; 72°C 10'; 4°C hold
• As proof reading taq I use the AccuPrime proof reading high fidelity taq from Invitrogen according to the manual
• Concerning the taqs etc. - I have made the experience that some fragments do not amplify with one taq but nicely amplify with another and vice versa so buying a different taq may work
• if you are amplifying GC-rich DNA you may want to add  5% DMSO (final concentration)

Ok, that's it right now.

Hope it's helpful.

Best and all the Best for 2016,

Christiane

Hello Isha Pandey,

Maybe you should increase the dntp, primer and taq Polimerase concentrations.

How long is the extention time you are using? The polimerase usually amplifies 1000bp in 1 min, so if you have a 800bp fragment, you should consider one minute of amplification. 

I suggest you to test six PCR reactions:

1) Increase only the dntp concentration and use the same machine program you are used to.

2)Increase only the primer concentration and use the same machine program you are used to.

3) Increase only the taq concentration and use the same machine program you are used to.

4) Increase  the dntp and primer concentrations and use the same machine program you are used to.

5) Increase the dntp, primer and taq concentrations and use the same machine program you are used to.

6) Use a different  machine program considering that in 1 min the taq polimerase amplifies 1000bp and do not chance the reagent's concentrations. 

Then, you can modify your protocols according to the necessities.

Good luck in your experiments!

Best,

Bárbara 

i would try increasing the annealing temperature until one amplimer fails and hope it was the shortmer. Also you could try adding betaine at final concentartion of 1Molar. Redesigning your primers or making a nested primer inside of your outer primer and re-amplifying  the firdt round product msy work. as might touch down pcr where you start with a too high annealing temperature and step down the annealing temperature to about 5c below the primer annealing temperature.

Do you need to get rid of the small band really?.

You can purify the large band from agarose if necessary or if the presence of the large band is diagnostic then just ignore the small one

Hi all,

Thanks a lot for your valuable suggestions, i got an amplification.. and i would surely apply all your suggestions in future.

"There I am getting two bands one at 400 bp which is non-specific and another one at 858 bp which is specific" this indicates nonspecific binding of the primer....so design gene specific primer having nucleotide length of up to 25 bases and increase annealing temperature (refer to RACE cloning manual for designing of gene specific primer) and try out touchdown PCR or gradient...Hopefully, this will give you the desire result.

There may be three possible ways to get specific amplification:

To summarise the issues mentioned by others here

1. To exclude the gDNA quality issues: Try performing a preestablished PCR from these samples or if none-exists- retry with reprecipitated /DNA (use not more than 40-100ng/PCR)

2. Use Hot start polymerase, if u are not using one already (In one of our genotyping PCRs, when we shifted from HotStar Taq ro TopTaq, we started having too many non-specific bands and had to reduce the primer concentration and increase the annealing)

3. As Sudhanshu mentioned above, Mg could be key and I feel this more for genomic templates. In one case, we had black and white results +/- extra Mg (with hotstar taq-Qiagen)

Good Luck 

• Following are some parameter that decides desired PCR amplification
• The quality of template DNA since it was amplified in one PCR but not in next. Confirm the quality of DNA by PCR with other current and any known set of primers for confirmation of amplification. If control reaction gets's amplification then template quality is good but your primer quality is at doubt so design new primers or try higher annealing temperature to reduce non-specific band and increase sp. band intensity
• Other options are increasing template or decreasing primers concentration will solve the problem of nonspecific amplification.
• Good luck

Hello Sachin Sir,

                            I had faced same problem. To troubleshoot that what I did was two step PCR. Add first 5 cycle amplification as follows: Denaturation (94 C for 30 sec), Annealing (50 C for 5 seconds) and extension (72 C for 50 seconds) and then follow specific cycle for your gene of interest. Hope you will get it. 

Dear,

Have you tried to design new primers for your gene? Have you checked if your species is the same as from the one the primer was designed? Have you tested gradient PCR? Best,