Hello,
I am trying to PCR amplify a 4kb gene sequence from a vector. The primers used are bit lengthy (>40bp). I have tried 20 different annealing temperatures from 50 to 70 deg C, I have even tried a 2 step PCR (skipped the annealing step as the Tm was 72 deg C) with Phusion polymerase and 5X HF buffer but failed to get amplification at any of the Tms in that range. The same conditions gave successful amplification with Taq DNA polymerase and Standard Taq buffer at a Tm of 62 deg C. However, since the next step would be Gibson Assembly cloning which again requires Phusion HF DNA polymerase, I am skeptical whether it will work. Kindly help me troubleshoot the PCR amplification by Phusion polymerase. Also, kindly let me know if Taq DNA polymerase would work well for Gibson Assembly.
-Thanks
Hi Sagar Kittur ,
When I used Gibson Assembly (quite alot) we frequently had primers that were 20bp for the annealing but contained 40bp overhangs. We used Q5 High-Fidelity 2X Master Mix (NEB, M0492L) following the manufacturer’s instructions. And had great success. And we followed the original Gibson Assembly protocol pretty much exactly with great success (90% gibson assembly success rate). The main thing is to make sure you are following the ideal conditions for the first PCR step as well as the Gibson step. We did not PCR purify the PCR before Gibson Assembly (found a higher yield without). Our Methods can be found Article The regulatory control of Cebpa enhancers and silencers in t...
Hi Shawn Krueger ,
Thank you for the prompt reply. I am a little confused here, did you guys use the Q5 High-Fidelity 2X Master Mix for PCR amplification of the vector backbone and the insert? If so, what is the error rate (in case you confirmed it by sequencing). I am facing a problem in the initial stage where I am unable to amplify the insert as well as vector backbone by Phusion HF DNA polymerase. I am using using primers of >40bp size (~20bp annealing and ~20bp overhangs). The reason I am sticking to Phusion is because of its lesser error rate compared to Taq polymerase.
-Thanks
1) Phusion polymerase has proof reading properties, meaning that there will be less or no errors during amplification. We had tried amplifying up to 3kb without error. Normal taq polymerase do not have proof reading properties, therefore, they produce higher error.
2) Check the quality (presence of inhibitors) and quantity of your DNA. For Phusion polymerase, the maximum DNA for a reaction of 50ul is 250ng.
3) Before amplifying using Phusion polymerase, you can check the melting temperature of the primers that you are using at this website: https://tmcalculator.neb.com/#!/main (Note that you only need to insert the part of primer that anneals to your template. You don't need to include the overhang part of the primers when calculating the melting temperature)
Hi Sagar Kittur ,
For our Vector backbone, we had that plasmid already in lab, so I would conduct a double restriction digest to linearize the plasmid in the Gibson Assembly site. For the insert fragment we used Q5 HF to amplify up the DNA sequence. Our success rate with Q5 with sequencing and RE screening was over 95% and our success rate for Gibson Assembly was over 90%. So from our experimentation the 60bp primer (20 bp annealing to insert and 40 bp overhang with the vector backbone) had the highest efficiency. We had 95% success with 60bp primers versus about 80% success with the 40bp primer design. Also with the primers for GA, we used the annealing temp of only the 20 bp annealing region and ignored the predicted Tm for the entire oligo. Hope this answers some of your questions
Hello Shawn Krueger ,
Thank you once again, yes it did clear a few doubts we had.
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