After a transformation into E. coli containing specific gene insert into plasmid vector pMV261, I again subject plasmid to PCR and could observe multiple bands along with specific band size 833bp. the positive control is the genomic DNA of M. tuberculosis subjected to same PCR cycling. PCR was done using Hotstart taq PCR mix from Takara. How should I trouble shoot this? conditions: 98°C-30sec; 68°C with grad till 71°C-1min; 72°C-1min, final extn 72°C-2min.
You should run the pcr with control samples of both empty vector and also bacterial DNA. This may account for some of your pcr bands. Also run an annealing temperature gradient or alternatively a gradient of DMSO from 0 to 8% final concentration which may clean up your amplifications using less primer as you have a large primer dimer amplimer at about 50bp
In addiiton to Pauls comments I would suggest to check the amount of plasmid used again. Keep in mind that 1pg of Plasmid is already a massive number of copies
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