I want to fuse a fragment with 600bp length to a 2.4 kb fragment with very stable secondary structures.
Overlaping primers have 42 bp length (54.8% GC content and total Tm=69ºC), their 3’ end (20bp) have Tm about 56 ºC.
End primers have cut sites and 35bp length (about 50% GC content and Tm= 62ºC).
Firstly, I have amplified small fragment and obtained specific band. Its unpurified PCR product is 470ng/µl.
Secondly, I have amplified long fragment with PCR master mix. Its PCR has specific band and some unspecific bands that I have extracted specific band from agarose gel. Its concentration is 13ng/µl.
Then I diluted small fragment to1/160. At the next step, I did SOEing PCR as attached file (slides 1 and 2).
As it is visualized in attached figure (slide 2), there was not any expected band with a size about 3kb. The small fragment has been amplified probably due to unpurified small fragment together its specific primers that were applied.
Could I use this final PCR product for next PCR with end primers? If it is rational, 1 µl from this PCR would be enough as template or diluted samples from this product are better? Should I purify this PCR product and then use it for next PCR. For next PCR, I want use Phusion HF DNA polymerase enzyme with DMSO (NEB). I would be very thankful if you help me to solve this problem.
Is there any possibility to decrease the lenght of the large fragment by a unique restriction site? I have a lot experience with SOE and the apoE gene, which is very GC rich, but its cDNA is only 1200 bp long and I always tried to avoid a SOE PCR using the complete cDNA.
I fear that an initial annealing temperature of 51° is too low for a DNA with extreme stable secondary structures, even if you are working with DMSO.
Perhaps you should first improve the conditions of your first PCR for the long fragment because even in gel purified fragments contamination occurs. Good luck!
I haven't had a lot of luck with SOEing in general. What I have had a lot of luck with is amplification of the entire plasmid and digestion with dpnI to remove methylated DNA template.
for me HF from NEB doesn't work for Over-extention PCRs, so I use OneStep Taq (NEB). I would run PCR1 and PCR2 on a gel and purify the products. and set up the reaction 3 as follows:
buffer 5ul
dNTP (10mM each) 0.5ul
R' (10uM stock) 0.5ul
F' 0.5ul
Long and short products (mol:mol =1:1)
Taq 0.2ul
h20 up to 25ul
PCR program:
1.94C 3min
2.94C 1min
3.50C 1min
4.68C 1min/kb so approx 3.5min
repeat 2-4 35 times
68C 7min
4C hold
hope this helps.
Alternatively, you could try joining your two fragments together using Gibson assembly (NEB), InFusion (Clontech), or NEBuilder Hifi DNA assembly (NEB). I always use Gibson assembly for inserting several overlapping fragments into a digested vector backbone and this works incredibly well. This is also very quick.
I have had good luck with SOE, and usually excise the bands from the initial step from an agarose gel and purify the gel away in the same day. I then add equal moles of each purified product to my second, SOE reaction with the addition of the outside primers (only add those primers) for 25 cycles. I usually design my template specific portion of the SOE primers so they have a melting temperature of 50C, and I use 50C for the annealing temperatures. In your case you could use an annealing temperature of 55 or 56C. If this doesn't work, or if you get a lot of non specific bands, you could try doing a couple of cycles with an annealing temperature of 56 and then raise the annealing temperature to 60C for the remaining steps. Also, make sure that your elongation times are long enough. You could try making the elongation 180s rather than 150s.
Best of luck!
I hope this helps… I was facing a similar problem where i was trying to ligate a 587bp insert to a 9kb long linearized plasmid with 43bp overlapping regions. So this is what I did:
Also, have a look at Inverse Fusion PCR paper by Markus Spiliotis. It might give you more clues for your work
Thanks for all your very useful information. and helpful advices. I would apply your suggestions to have a graet result.
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