I am attempting to amplify a 15.4kb sequence from genomic DNA. The DNA template is high quality (fragment size >80kb) and I'm using the Generuler HR ladder. Gel is 0.4% agarose. I'm using the enzyme Herculase with 4% DMSO. I designed the primers with primer3 and checked for specificity using UCSC in silico PCR and Primer-BLAST.
Cycling conditions:
Initial denaturation: 92°C, 2 min
Denaturation: 92°C, 20s
Annealing: 63-68°C, 20s (gradient in image)
Extenson: 68°C, 8 min
repeat 10 cycles as above, then 25 cycles adding 20s / cycle to extension (so 8:20, 8:40 etc.), so 35 cycles in total
Final extension: 68°C, 8 min
There are 2 bands that approximately correspond to my expected length, but there's also a 20kb and a 24kb band and shorter band (~10kb). The top annealing temperature of 68°C (5th lane in image) looks best in terms of specificity.
How can I eliminate the unspecific bands? Also, I'm not sure that my product has got the correct length. The template contains many repetitive sequences and GC-rich areas. How can I avoid formation of secondary structures?
Manfredi Carta
what are you going to do with 15kB DNA piece?
Sequence, subclone, or something else?
Oskar Laur the 15kB DNA piece will be used for long read sequencing.
Ruslan Kalendar why do you recommend higher annealing temperatures? The recommended extension temperature for templates >10kB is 68°C for Herculase II Fusion, the enzyme I'm using, so I assume that any incorrect sites to which the primers wouldn't anneal at annealing temperatures >68°C would be bound during extension?
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