I have six constructs with the following G-C contents: 29.6%, 23.2%, 27.2%, 24.4%, 27% and 28%. Due to the low G-C content of genes of interest, the Tm of my primers (see attached file) are low; calculated between 45 and 55 °C using the Thermo Scientific Tm Calculator (Taq-based).
I started off with a gradient PCR using the protocol slated below for all six constructs. I had no product but primer dimers appearing as a thick band below the last band of a 1 kb plus ladder. At the end of the day, I had optimized annealing temperatures between 38 °C and 65 °C (First 38-52°C and then 48-62 °C). I played a little with the extension time (increasing) but no luck
Component 50 μl rxn Final Conc.
10X DreamTaq Buf. 5 μl 1X
2 mM dNTPs 5 μl 200 µM
1.25 µM Primers mix 5 μl 0.125 µM
Template DNA 1 μl 2 ng
Taq DNA Polymerase 0.5 µl 2.5 units/50 µl Nuclease-free water 33.5 µl —
Cycling conditions
Step Temp Time Cycles
1. Initial Denat. 95 °C 2 min
2. Denat. 94 °C 30 sec
3. Annealing 38-52°C/48-62 °C (Grad) 30 sec
4. Extension 68 °C 4 min
5. Go to 2 24X
6. Final Ext. 68 °C 10 min
7. HOLD 4 °C —
I increased the Mg2+ conc. and nothing happened. DMSO won't be my friend because of the low GC content I guess :).
I checked the conc. of my ordered genomic DNA to 130 ng/μl on the NanoDrop. I decided to increase the concentration by adding up to 100 ng/50 μl Rxn and nothing. I have read here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145803/ that reducing the extension temperature (to as low as 60 °C) required for PCR amplification of extremely A+T-rich DNA leads to product formation. That did not work. Instead, I had some unspecific product for two of the constructs as well as primer dimers. I have also done a control experiment together to validate the reagents and everything worked very fine with the control. My primer sets (forward and reverse) are mixed in a single-stock aliquot. Could this be the problem?
Please I will greatly appreciate any proposals. Thanks in advance.
Even I am struggling with the same problem. I have read protocols online and got amplification (Non-specific bands also). I tried repeating the PCRs to get enough product for cloning, but then got very little and then almost no amplification. I have tried MgCl2 gradient and got results at 3M concentration. The PCR ramp time can also play a role. So it's better to try different annealing and extension times. One online post has mentioned using just 2-step PCR. No separate annealing step. One paper mentions getting the best results with Phusion enzyme. Ordered it. Will try and let you know the results. I have attached the screenshot pdfs. Please do share if you get any success. All the Best!
Even I am struggling with the same problem. I have read protocols online and got amplification (Non-specific bands also). I tried repeating the PCRs to get enough product for cloning, but then got very little and then almost no amplification. I have tried MgCl2 gradient and got results at 3M concentration. The PCR ramp time can also play a role. So it's better to try different annealing and extension times. One online post has mentioned using just 2-step PCR. No separate annealing step. One paper mentions getting the best results with Phusion enzyme. Ordered it. Will try and let you know the results. I have attached the screenshot pdfs. Please do share if you get any success. All the Best!
How long are your primers? Using long primers might help...Good luck...:)
i agree with Harinder. Long primer also Using a higher annealing temperature might help
There are a bunch of other things you can do, but longer primers will help greatly with specificity and your TM. Unfortunately, they do little to affect your product yield if the actual amplicon is repetitive, low complexity, or extreme AT/GC percentages.
On a side note, nope, mixing your primers is unlikely to affect it, unless they have significant heterdimer structures (IDT has a predictive tool: https://www.idtdna.com/calc/analyzer)
I assume from your rather long extension time that your amplicon is ~4kb? Long amplicons like that require very well made genomic DNA. Have you run your template on a gel to make sure you have long pieces? I've had excellent success with long PCR, between 10-5 kb when the genomic DNA runs as a fairly sharp band about the 23kb ladder. You'll want to pour a .5-.7% gel and run it slow to allow for separation.
If the DNA is indeed long and intact, I would definitely suggest trying Phusion, or if you can get it, NEB's Q5 polymerase. In addition, to continue to toss out potential solutions, I usually create a matrix for long/difficult amplicons where I modulate buffer concentration (1x, .75x, .5x), betaine, 1,2 propanol, dNTP concentration, and finally polymerase amount. The greatest differences I've seen personally, are changing betaine amounts, and altering polymerase amount.
If your DNA is not doing so well, I highly recommend trying out this old protocol I found a while back: "Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques" by Salah Aljanabi and Iciar Martinez, Nuc Acids Res, 1997. They have a representative gel image of what your genomic dna will look like, and whenever my template has looked like that, I have eventually gotten my product.
Hopefully something in that mess becomes useful for you, and good luck!
Longer primers....IDT make primers greater than 60 bp (ultramers). Also my favorite kit for one off PCR cloning needs (as in you only need the product once) I use epicenter MasterAmp kits, especially the extra-long kit, It comes with 9 buffering conditions and I just about always get a product in about ⅓ of the reactions. It uses a combination of 3 polymerases including Taq, so some of the ends of your PCR product will have an a-tail. Just add some more taq/ATP and incubate at 72 degrees after PCR and sub clone into a t-tailed vector (my favorite is the invitrogen PCR2.1)
Also another thing to try is a slow ramp from your annealing to extension, this gives the polymerase a chance to elongate from your primers, increasing their Tm.
But, the best thing to do is remake your primers.
Good luck
Glycerol , DMSO and other additives might help in the inc of reaction efficiency.
This is a standard problem in cloning promoters from eukaryotes. Google "genome walking" or "genome walking kit", you will find protocols designed for AT-rich templates. You might have to invest in a special Taq as well (at least for me, what worked with the Taq from a Genome Walking Kit did not necessarily work with the standard lab Taq). Good luck!
Hello all,
Thanks very much for your kind responses. I have tested some of the recommended procedures highlighted. I increased the the [MgCl2] to 8mM and varied the genomic template amount by 10, 30 and 40 ng. i obtained the results on the gel picture attached.
In lanes 1, 2 and 3, a 192 bp fragment was expected while in lane 4, 5 and 6 a 558 bp was desired. The desired band for lane 4, 5 and 6 seems to be okay though very faint and also accompanied side products. But that of 1, 2 and 3 seems to be non-specific bands since they had to appear under 200 bp on the ladder. I have read somewhere that short pcr products might migrate differently on the gel, as well as dimerization of products. The band directly above the approx 300 bp band in lane 1, 2 and 3 appears to be doubled its size. Could this be the case? Are there other propositions that could help improve these results?
Thank you very much.......
Hello everyone,
thanks very much for all your comments that enabled me to make a lot of findings to pinpoint the source of my PCR problems. I finally sorted out that I had a template problem. I ordered the wrong genomic DNA with information obtained from GB. The strain had a patent reservation and so it was removed from public strain collection. However, in my reference paper, the authors had mentioned it as the wt strain. This was the cause of my nightmares. Nevertheless, I contacted the group responsible and got the right strain and everything is working now well :) (see attached image)
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