Hello,
Does anyone have experience with LD-PCR implementation and optimization? I have been trying to implement a LD-PCR protocol in our Molecular Biology lab, but it seems it does not amplify anything.
Shaima R. Banoon - thank you, I will check this publication.
Göran Hjälm
- Thank you for your interest! We are using genomic DNA extracted from peripheral blood leukocytes. The expected product size is 10-12 kb.
Göran Hjälm
- thank you for your input, your comments are extremely valuable nevertheless.
I have tried and optimized amplification of 3-4.5 kb. While optimization, I prepared a master mix with DNA in large volume and aliquoted into different tubes. Then, I run PCR experiment in three different thermal cycler with same cycling condition. I got better amplification in thermal cycler with slower RAMP rate. Theoretically, slow means smooth, smooth means fast amplification and extension.
Make sure that your master mix and enzymes with appropriate additives to support long range amplification. Please provide sufficient concentration of dNTPs and MgCl2. Most importantly, reduce ramp rate in thermal cycler.
Recently, I saw a paper regarding the impact of ramp rate https://www.nature.com/articles/s41598-018-21458-y.
Narayanan Kalyanaraman - thank you for your valuable comments.
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