The future expected problem -
So, I plan to join two DNA fragments A and B by overlap PCR. Amplify individual fragments use them together as template and then amplify the combined fragment ab using forward primer of A and reverse primer of B.
A - 1.1kb
B - 1.3kb
I expect a 2.4 kb fragment in total if overlap PCR works. But, I have a feeling it won't work.
Previous experience - 1
Fragments A and B are amplified with such they have Xba1 sites in their reverse(A) and forward primers(B) respectively. Then I restricted fragment A and B with Xba1 to produce sticky ends. Then I ligated them (50ng each fragment). When I run the ligation mix on a gel, I get a fragment of the right size 2.4kb along with other bands. I cut out the right size. However, I am not able to use this 2.4kb AB fragment as a template for PCR. I cannot get it to amplify. Any tips on how to amplify?
In the previous situation, one can argue that gel purified ligation mix yielded only very low amount of DNA, so perhaps that's why no amplification
Previous experience - 2
But, I also have not had success using gel purified PCR fragments as template for PCR before. These are in good concentration after PCR and gel purification, around 70-80ng/ul. But I still cant get reamplification using them as template. It seems to only work when I amplify fragments cloned into plasmids.
So any tips?
Before I invest in primers, I need to ensure that if my overlap PCR works, I can amplify the product further using the gel purified fragment (AB) from the overlap PCR.
So, 1 tip, I got from reading through similar questions is to dilute the template. If so how much? I don't understand the logic in this because, I sometimes use 1ul isolated plasmid around 250ng for PCR amplification, which is a 6kb plasmid construct with 1.5kb gene(target of amplification). So that means that there is at least 62.5ng of templated DNA for the PCR. This is pretty close to the 70-80ng/ul concentration you get after gel purification and elution of fragment from PCR mix.
So I am at a loss. I use Thermo phusion polymerase for amplification and Thermo fast digest for restriction.
Hello Rohit,
I have recently did the experiment where i use PCR product as a template. In this situation i had good product when you dilute the template (PCR product) to 1:50 or more. In theory it should be more diluted sometime to more then 1:100 or more. I can suggest you to have some parallel with dilution start from 1:50 and higher for template in your PCR reaction. I read that if you use undiluted or less diluted template (PCR product) for reaction, in sometime inhibit the PCR reaction because of high concentration of template. So you can try to work with some higher dilutions. Good luck.
Best,
Mradul
Mradul Mishra Thanks for the answer! I'll give this a try. I have a few more questions if you don't mind. Were you also trying to amplify from assembly PCR or just a normal PCR? Also which polymerase are you using? Size of the fragment? And concentration of template after the first PCR?
Rohit James Your welcome. I was just performing a normal PCR for cassette amplification, but i needed more amount (concentration of Product) for my further experiments so I did the two steps PCR. The product size was 3kb and I used phusion polymerase. I do not remember the exact concentration of my product after 1st PCR (very weak band on gel) but I am sure it was less than 100 ng/ul.
Mradul Mishra Right so.. basically considering 100ng/ul as the original.. so 1:50 dilution would mean around 2ng/ul. Alright thanks Mradul Mishra . I will try this.
Massive dilution really may help. Also, consider using Fusion PCR protocol (four primers in reaction, "inner overlapping "primers at much lower concentration, can be googled). This is an easy one step (can be done in separate reactions too) protocol, with generally good results. Martin
Dear Rohit, you can certainly do it, but i suggest to you to use an high fidelity polimerase ad
KApa Hifi (roche)
CLone amp (takara)
Pfu utlra fusion II (Agilent)
Q5 (NEB)
those work very well at high template dilution (generally i'm using 0.1ng template in 25ul PCR reaction)
because each PCR has a certain probability to generate unattended mutations and with a stantd Taq polimerase you risk to obtain a mutated clone at the end of the 2 PCR.
good luck
Manuele
Martin Lenicek Thank you! Any idea why the low template amount works? Because PCR normally generates microgram levels of DNA.. so I think within a few cycles it would easily reach 100ng. And yet when you amplify from a plasmid template it doesn't seem to matter. But if I you start with 100ng of a PCR amplified gel purified template its a problem. Any explanation as to why?
Manuele Martinelli Thank you! This is good practical advice. I have both Phusion(Thermo) and Q5(NEB) available in the lab. Both are high fidelity polymerases.
Also you mentioned using 0.1ng for 25ul reaction. In this case are you using PCR amplified gel purified fragment as template or amplifying from genomic or plasmid DNA?
Dear Rohit
0,1ng in 25ul,refer to plasmid DNA. If you are using gel purified PCR, probably if the PCR size is small you can also use less amount, but i do not think that it will make a great difference.
Manuele
@Rohit Gel isolated PCR is often tricky business (even if you are sure, you ve cut the right band - this may be challenging too, as mobility of ligation mixture may be slightly different from the marker and you may not be able to distinguish A-A, A-B and B-B ligation products)- unspecified amount of interferents (polyssaccharides) - massive dilution would dilute those. Without PCR purification you are working with multiple wrong products, lowering your chance even more. If you can, redesign your inner primers to heve larger overlap (20bp) and run fusion PCR. This is much more efficient than joining the fragments after restriction.
Martin Lenicek Thanks again Martin. I've seen a couple of fusion PCR protocols. Can you please share the one that you use? Specifically one that details the amount of DNA of each fragment to be added?
Rohit James Hi Rohit, template conc should be optimized. I suppose, you are going for two step (you already have both PCR products). You can check m protocol attached.
One step would be similar - template concentration as in regular PCr, four primers in the reaction, inner (overlapping) primers at limited amounts (should be optimized). Typically, I serially dilute inner primers (5x, 25x, 125x, 625x, 325x) and check, what works best. Good luck Martin
For my gene deletion, I PCR amplified two fragments, gel purified and tried to join both fragments using overlap PCR. After several attempts with no success, I tried a regular PCR (Q5 polymerase) with terminal primers using 1 ul of each purified fragment (80 and 140 ng/ul) and boom I got a result.
As mentioned above, you could redesign your inner primers so that overlap region would be at least 21 bases to enhance joining of the two fragments. You could also try diluting PCR products to 1/10, 1/100 before use for overlap PCR.
Hi Rohit James,
I have had a similar experience in the past; not being able to use PCR products as templates for another round of PCR. I can't remember very clearly, but I think the problem was at a 3rd round of PCR. Many hypotheses were suggested as potential causes of this. But back then, while trying to figure out what was wrong, something I found quite interesting was the "shortening" phenomenon discussed in the paper below:
Article Construction of long DNA molecules using long PCR-based fusi...
However, I never had such problems again since then, and overlap extension PCR (OE-PCR) now works so well for me and has in fact become a routine. Below is a simple one-pot OE-PCR protocol that you may follow.
Article Gene splicing and mutagenesis by PCR-driven overlap extension
If you wanna experiment more (with split OE-PCR), you may check the following as well.
Article Rational-Based Protein Engineering: Tips and Tools
Article Overlap Extension PCR: An Efficient Method for Transgene Construction
The (split OE-PCR) procedures described in both are very similar, but you might find recommnedations in there to help you optimize your protocol. Still, the first one-step protocol above works well for me. In addition, I suggest that you use equimolar (pmol) amounts of the fragments instead of the ng concentrations stated in the protocol.
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