Can anyone help with long range PCR optimization?

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No title

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MiRNA qpcr problem?

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Why do the PCR bands from the cDNA templates (extraction from the cell after transfection) show different size from the band of the plasmid template?

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Gregory Dressler

Do you mean Next generation Sequencing? You really do not need to do NGS to sequence a 10 kb fragment. NGS requires making a library and will generate millions of short reads that must be assembled and aligned. You would get a more accurate sequence by conventional methods using primers across the region.

Sathiya Maran

Hi Gregory Dressler,

Thanks for your comments.Yes next generation sequencing. My target gene is 40kb so i have broken it into 4 amplicons with each amplicons 10kb. one of the amplicon is the one with faint band? so can i proceed with NGS?

Sabitha Omprakash

Try with LA TA KA RA Master mix. Use 50 -100 ng of DNA, run with TAE buffer.

Amr Halawani

Sometimes it is worth to reduce cycle number to 20-25. Use a polymerase such as TAKARA PrimeSTAR GXL will help to give you a clear products. Double check with primer design in which must be specific and not picking up other targets. I hope that helps.