Dear all ,
I need to amplify, clone and express 657bp of viral RNA in bacterial system . I designed gene specific primers with buffer sequence, restriction sites and the complementary sequence along with start and stop codon. First , I isolated the RNA and got value of 2.02 for 260/280 in nano drop and used the same for cDNA synthesis ,, then diluted it to 5 times for PCR amplification . Initially i tried in 58 and 59 degrees but i did not get any amplification. Then, tried at 53 degree for 32 cycles (with different dilutions of cDNA) and I got the amplification of 657 bp product but with non specific bands so i increased the temperature to 55 but i did not get any results for it ,,and i tried gradient too from 45- 53 degree but i haven't got any results . one thing is that there was spurious amplification of nearly 100bp- 120bp fragment in all the temperature profiles i worked out and bit confused with that. can any one help me with this issue. and suggest me the annealing temperature
i have herewith attached the gel pics of RNA, cDNA and the amplification at 53 degree with 32 cycles , 54 degree with 4o cycles 58 and 59 degree.
GCCTGA GGATCC ATG GACAAATCTGAATCAACCAGTG (UNDERLINED SEQUENCE IS COMPLEMENTARY)
GCCTGA GAGCTC TAA TCAGACTGGGAGCACTCCA (UNDERLINED SEQUENCE IS COMPLEMENTARY)
thank you in advance