I amplified several PCR products and the bands were pretty good in size and unique. The PCR products were inserted into pENTR/D-TOPO, then they were transfromed into E. coli (TOP10 and DH5 alpha). I checked the colonies in PCR (solid-colony PCR), which also gave the expected bands (plasmid specific forward and gene specific reverse primers. I passed these colonies into liquid LB and the cells grew in culture. After doing miniprep, there was no DNA extracted at all, like if the bacteria had lost the plasmid while growing in the liquid LB. We PCR the liquid LB-grown colonies, and there was a very shallow band (much less intense than in the solid colony PCR). The resistance of pENTR/D-TOPO is kanamycin. I used the kanamycin in other transformation, it worked so we didn't think it was a wrong preparation of kanamycin. Does anyone know why plasmids are lost in liquid LB? We had used two different kind of the Miniprep kits and they worked in other plasmids. So I didn't think it was the problem of Miniprep kit. Could someone help me exlpain the problem? Thanks!
Chen Zhang Did you grow the colonies on LB broth+Kanamycin? If yes, how well did the strain grow? could be the kit? did you have manage to isolate any other plasmids at the same time using the kit?
Kiran Sreeja Jayan Hi, Kiran, thanks for your answer!
Yes, the starain grows vey well. I used two different kind of the Miniprep kits and they worked in other plasmids at the same time. So I didn't think it was the problem of Miniprep kit.
Hi Chen Zhang, did you solve the problem of having a faint fragment ?, I have the same problem in the miniprep PCR
Yes, we already solved this problem. There was a problem in pENTR™/D-TOPO™ Cloning Kit. We just changed a new pENTR™/D-TOPO™ Cloning Kit which was produced in different batches. I hope this answer will be useful for you. Javier Alburqueque
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