trouble in plasmid pcr - i cannot obtain designed band from a definied plasmid (Jul/08/2002 )
i cloned a gene into pGEM-T easy vector(Promega) and sequenced it. then i designed a pair of primers according to the sequence result. i use PWO DNA polymerase(Roche). but i cannot get a single band. even the main band of the designed MW is not what i want. pls tell me what my problem is. thank you.
Can you PCR using SP6 and T7 oligo to ensure that the template is able to be PCRed from? This will also tell you the size of your insert.
First,you can make a cut in template to make PCR easier.
second,try to PCR in two annealing temperature combinations: such as 94 20'' 56 20'' 72 50'' 4X
94 20'' 59 20'' 72 50'' 32X
I have had multiple band problems in the past which were easily resolved by linearising 1ul of the plasmid (from miniprep) in 10ul with a single restriction cut, then putting 1ul of this into 15 cycles of PCR with low concentration of primers (I use 2uM stock and put 2.5ul of each into a 25ul reaction). There's loads of DNA so 15 cycles is all you need and lessens the chances of getting multiple products later in the PCR. Plus if you have primers at too high a concentration, they are also likely to give multiple products. Hope this helps.
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