Hi , For the past 2 weeks I have been struggling with something so simple. I have a plasmid ( vector is pcDNA4/TO with amp resistance). It has worked perfectly before and recently I was close to running out of it so I did the transformation.. had colonies so I seeded the cultures for maxiprep or miniprep. After O.N incubation everything looks ok so I proceed with the plasmid extraction and for some reason my yields are terrible( 50ng/microlit and the 260/280 ratios are not that good either). I have tried different kits as well as tried re-transforming with the original batch. In parallel, I also transformed another plasmid which has amp resistance and the minipreps for it worked just fine. It is only this one which is causing a problem and I don't know where to start the troubleshooting. Unfortunately no one else in the lab is facing a similar issue so I am unable to get any ideas from them.
Any inputs would be highly appreciated.
Hi, I have a question. You talked about mini and maxi, did you have the same low results also with miniprep? About your original batch, did you check the sytatus (load on gel after restriction) of the batch (I presume was a previous maxi prep) and when you said that before worked perfectly, did you have an amout of this maxiprep that worked fine? It is possible that you have a problem with this plasmid preparation, for this reason I prefer to have not only a batch of plasmid extracted (maxiprep), but also an aliquot of bacteria in glicerol
Hi, Yes I checked the original maxiprep and miniprep stock which I have made and those yields were great. 2microgm/microlit. The 260/280 ratios of those stocks were good too and I have successfully used that batch for my experiments and had good results.
Hi, from my experience I have problem with a maxiprep, perform a restriction and load onto gel, the maxiprep it is not very good and caused me problem with trasformation. but if it is not the case i would try to trasform again but from the miniprep (the good batch), in gereral less concentrated, more cleaned.
You can check also you bacteria growth after ON, sometimes with mini as maxiprep a lot of initial material is deleteriuos because clog the column and gave poor results (it is happened to me, I am so happy for the bacteria growth but not so happy at the end); you can seed to perform 5-6 miniprep and then make a pool.
Hi Zankruti,
OMG, this is the biggest mystery of my life too! I also faced the same problem a couple of times and the strange thing is that, it sometimes works and the other times doesn't! and this is while I subculture from the same stock every time which I keep at -80. anyhow, since i myself still don't know the exact solution to this question, I believe it helped me when I kept the bacteria for growing less hours than usual (around 10-11 hours) and it seems that after some point the more they grow the less plasmid yield i get by the end.
Also, my constructs have Amp resistance as well! (in case this can lead to any clue)
Anyhow, I have tried so many different brands of mini prep kits for solving this problem, but to this date i still don't think the problem comes from the kit. I would be so happy to find out the answer to this question as well.
Cheers
-Shirin
Hi.. to update.. the technician had changed the agar being used for the plates.. the agar was softer and for some reason, I don't know why, but when we switched back to the previous way of making the agar plates.. things went back to normal :)
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