I want to amplify the 5 prime upstream sequence up to 2kb, of genes, after several attempts i did not got specific primers or some suitable amplifying condition to get that sequence. Please anybody can help me in primer designing and PCR amplification
in this regard
is the genomic sequence of these regions already known or are you looking for unknown sequence?
If already known online try smaller fragments eg 1kb then a further kb rather than all in one amplimer
If the sequence is unknown you may want to look at inverse pcr using known gene sequence
the sequences of these genes are predicted genome browsers and we want to authenticate and cloned these for finding core promoter region of respective genes
I would move the primer positions of the non coding primers in case your primers are sitting over polymorphisms and check if the amplimers are high GC.If so you can run the pcr in 1Molar betaine or 1%-7% DMSO to get better amplification and try to make sure thata your primers anneal at similar temperatures and at 60c or higher. In your pcr reaction run a primer set that always works well just to check that the reagents and equipment are working well
Respected Paul, if i send these sequence to you can you help me to analyze this sequence and suggest me some more valuable things,
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