I am using Fermentas pfu polymerase. it is provided with single buffer vial which has MgSo4 also.
What is the amount of plasmid you are using for your PCR reaction? Maybe the difference is the amount of target DNA you are using. Remember that when using plasmid you should use a low amount of it (10 ng tops)
Try to use several amounts of plasmid DNA ranging from 1 ng - 100 ng to carry out your PCR reaction.
Also, I recommend to isolate your plasmid DNA with a Plasmid DNA extraction kit to avoid the use of phenol that inhibits the PCR reaction, and the use of an E. coli such as DH5 alpha or XL1-Blue by their low content of carbohydrates that might inhibit PCR reaction.
If you want to verify that your positive plasmid DNA control you may carry out a restriction enzyme assay to liberate your cDNA cloned or to generate a specific restriction pattern for your plasmid.
Regards
Thank you for the kind response sir, I am using 50 ng of plasmid DNA for PCR. But the same plasmid has been used with the Genei Taq polymerase which gives good & Exact amplicon. Now i am facing another problem that the same Taq polymerase amplifies the plasmid positive control but the cDNA has not get amplified. Please help me sir ! I am using cDNA derived from the final step of 5' RLM RACE protocol. I have tried with long DNA polymerase also. this polymerase doesn't amplifies the particular cDNA.
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