Hello researchers!
Now one of my work is assembling two DNA fragments with homologous arm into one fragments which as 2900 bp length. But according to the results of the PCR, the length of the product is almost only 900 bp, which is a big gap compared to the correct length. Coincidentally, its length was just twice as much as one of the fusion fragments. At present,I speculate that the reason why I can not get the correct fragment is the mismatch in PCR reaction. I have tried to increase the Tm value, but there seem to be useless. Is the disparity in the length of the two fragments important in the progress of fusion? How can I get to the correct product?
Looking forward to your answer and discussion!
Hi Liu Zhi Fei, this might be a problem with your electrophoresis because your fragments is large, let your ladder open more and reduce your gel electrophoresis percentage
Hi Liu Zhi Fei, can you please provide the thermocycling conditions?
I have done this overlapping PCRs and when I had problems like this, the reasons were:
1. Incorrect DNA quantification of the fragments to join. The ratio should be equimolar.
2. Try prolonging the extension step in your PCR according to polymerase manufacturers instructions or increase it slightly. For example, if manual says that 1kb/15s, try 1kb/20-30s.
3. Try different buffers or consider using additives such as DMSO, it decreases the annealing temperature.
4. If still no positive results, I would try first running a few cycles (10) without the primer fw from "PCR1" and primer rv from "PCR2". This will firstly join your two fragments without amplifying the joined product but you will have the whole piece to be used as template. Then, add primers to the tube and use a normal thermocycling program (with adjusted extension times) to amplify your joined fragment.
If you provide more details on how you did this and info about your starting material, it will be easier for us to provide some help.
best.
If the length of two fragements are different, then you can adjust the ratio. For example, if you use 450 bp and 2550 bp fragments, the ratio of the fragments in pcr reaction should be 1:5.6. ( for example, 1 ul of 450 bp and 5.6 ul of 2550 bp). How much concentration of fragments did you use for PCR?
In order to make the conditions more clear, I'd like to add some information.
1、The reaction was designed to obtain a gene with base mutation from Escherichia coli MG1655. After the purification of the genome of the strain, I checked the purity and concentration of DNA with Nano Drop.
2、The system of the PCR was set to 50 ul, which has 25 ul Primerstar Taq Polymerase，1 ul temple and F/R primers and ddH2O up to 50 ul.The time program was normal as we used.
3、I designed the primer again which differ from the old was the mutant site moved to the middle of the primer. I was wondering if the exist of the mutant site in 5' of the primer would make it difficult to overlapping, although it's easy to obtain it's separate fragments.
I don't really understand "The time program was normal as we used." Cycling conditions should be optimized for each reaction based on length of product, nature the template, among other parameters...
How many bp are overlapping between the fragments you want to join together? Normally I do 15-20 bp.
Can you amplify the two fragments with the point mutation successfully?
1ul of template is not very accurate, for PCR we should consider ng of gDNA. For my polymerase I use normally 100ng.
Like you said, the cycling conditions should be described specifically.
1、I use the program as 95℃ 10 min 95℃ 30 s 61℃ 30 s 72℃ 60 s and 72 ℃10 min，with the 30 cycle from step 4 to step 2. The reason why I choose this program was making sure that primer correctly pairing and product fully extending. The extend rate of Taq polymerase was 4000 bp/min and the length of my target fragment was 2900 bp.
2、At least, two fragments with the point mutation were amplified successfully. And their concentrations were 77 ng/ul and 114 ng/ul. The overlapping sequence's length came to 25 bp, short length of above sequence always made me worried about it's mismatching and unstable combination.
3、Balancing the concentration of two fragments before the cycling makes some sense, it would be tried in my further study.
Thanks for your advise and looking forward to more suggestions from you!
I think the PCR conditions look good. If you already have the individual fragments you want to join together it is just a matter of twisting PCR conditions by:
1. First, use a more proportionate ratio of the two fragments (equimolar amounts)
using using the new fragments you will get and see if you can get the whole fragment with your standard conditions.
2. Try extending the extension time for the overlapping PCR reaction as the specifications of the polymerase are based on average processivity of the enzyme over diverse templates. In my hands, my polymerase which processivity is 1kb per 30-60s needed 3 minutes to amplify a fragment of 2kb correctly, but not achieved if time was reduced to 2 min. So you might try maybe 90s or 120s for extension times if the PCR doesn't work with you standard conditions.
3. Check this paper, it might give you an overview of this process and things you could try if the PCR still doesn't work. Article Simultaneous splicing of multiple DNA fragments in one PCR reaction
Best regards and good luck!
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