Hi Friends,
I have to isolate the complete coding part of the selected gene which size is 2.5kb contain 12 Exons. Particularly Exon 1 (Starting bases) has GC content high as >70% (high energy secondary structure). Rarely I will get the amplification of expected size as faint band in Gel; try to reamplify the amplicion but ended in high density smears in the gel.
1. Any protocol specially for the gene has high GC at 5' coding end?
2. Did I want to modify PCR protocol for re-amplification of 2.5kb amplicion?
Suggestion welcome!!!
Thankyou
Ellango
Some suppliers of DNA polymerase (e.g. Phusion) provide a buffer that is designed for high GC templates, at the cost of a somewhat lower fidelity. However, if your purpose is to generate a clone expressing a recombinant protein, you may consider having the sequence codon-optimized and synthesized. In the end it may be cheaper than loosing your time to no avail.
with a high GC region I would run both the RT and the pcr in 1M betaine to assist easy melting of the high gc region. If you are re amplifying it is very easy to over amplify and get concatenation of the amplimer so very long amplification products. For re amplification I would dilute the band 1 : 100 and amplify 1ul for 10-18 cycles removing a tube every 2 cycles and one should give good and clean amplification. It might also be possible to amplify in 2 parts initially then ligate the 2 amplimers
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