I am working with gateway cloning and want to amplify a gene using attb primers (44 bp). I added the attb sequences to my primers and got synthesized from sigma. Now the problems are
1.The melting temperature is around 80 degree Celsius though the GC content is above 50.
2.It has some secondary and i can't change the primer sequences bz it is selected for amplifying the whole gene which is about 800+ bp.
3.I am getting a band near 100 bp 2 log ladder (New Eng Biolabs)which can a dimer (but I am not sure)
4.I am using this in wheat genomic and cDNA. Pcr conditions for 10ul rxn are 1 picomole primer,1.5nM mgcl2, and 60c annealing temp.
Please help me to solve this riddle. Let me know the rules to make attb primers
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Gateway attb primers not working
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