1. As Kiran Sreeja Jayan mentioned, it may be problem of PCR conditions. You should introduce positive control to be sure about PCR.
2. Sometimes it is hard to denaturate bacterial DNA (even with initial dentauration 10 minutes in PCR), you may try to boil bacteria sample first and then add to PCR mix.
3. Something could go wrong before (like digestion, ligation etc.) and you just did not obtain positive colony.
All the best!
Did you have a positive control to make sure your PCR was working? Do you know if your primers can work under the conditions you performed the PCR?
1. As Kiran Sreeja Jayan mentioned, it may be problem of PCR conditions. You should introduce positive control to be sure about PCR.
2. Sometimes it is hard to denaturate bacterial DNA (even with initial dentauration 10 minutes in PCR), you may try to boil bacteria sample first and then add to PCR mix.
3. Something could go wrong before (like digestion, ligation etc.) and you just did not obtain positive colony.
All the best!
Culture media in which the bacteria are grown can inhibit PCR reaction.Pick up single colony. Boil at 100 degree in 100ul sterile D/W for 10 min. Spin at 1200rpm for 5 min. use sup for your PCR. As others have pointed out your PCR reaction should be robust.
Hope this helps.
Shefali
Make sure your colonies are freshly grown and also do not obtain the entire colony, too much bacteria will not be beneficial and can inhibit the PCR reaction.
Dear Abbas, as other reseachers said that you should take a freshly grown single colony, it can work.
Sometimes in some bacteria due to extracellular products and some in sufficient digestion of cells, the amount of DNA will be low and high amount of proteins and other cell substituents, macromolecules can interfere with PCR rxn. For this reason, we prefer enzymatic digestion of freshly grown bacteria in broth.
Best regards.
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