Could you describe how you prepared the DNA for the colony PCR? addition of large amounts of the bacteria directly into the reaction tube can inhibit the PCR.
I first picked up the indivisual colonies using micropipette tips from the transformed plate and it was dipped in the pcr wells containing water, after that mastermix was added and then forward primer/reverse primer was added before proceeding for the reaction@Hanna Alalam
Hi, I used to have the similar situation with you. In my experience the DNA just get stuck within the gel (as you can see the signal around the well). Thus, the solution are
1. Pick less bacteria from the colony
2. Make sure that the reaction buffer is clean (if you use tip or toothpick to pick colonies , over fire may also interfere the results)
good luck
This is most likely a case of excessive bacterial colonies (you only need to pick a tiny amount since less is more in this case). Run controls with the mini-prep to make sure you reagents are working then repeat by picking a smaller amount.
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