I'm screening for my plasmids which are pT7-FLAG2 with inserts approximately 1000-3000 bp each. My plasmid is 4.8kbp. I've cultured them in BL21 and tried extracting them using Birnboim and Doly method. However, what I got was a huge white blob at the end of the gel. I then tried adding RNase but this wipes out all my bands. I'm baffled because even if I don't have my inserts, by right I should see a band showing my plasmids. Besides that, I reuse my reagents (alkaline lysis solution 1,2 and 3) but I store them at 4C when I don't use them.
Please don't take this the wrong way, but unless you are going to be doing thousands of minipreps and/or you cannot afford it, may I suggest simply buying a commercial miniprep kit? If this is not an option, try to find someone in your institution who has experience doing/troubleshooting homebrewed minipreps. Her/his advice will be invaluable and much more to the point than what we can offer from here. That said:
- BL21 is not good as a cloning host. Miniprep yields will be poor, the preps may have plasmid concatamers, and will require an additional step (phenol extractions, or silica columns are the two most popular choice) to get rid of nucleases. Try to find a recA endA strain (I use DH5alfa, XL1-Blue, SURE and STBL3 but there are many others. NEB has a good selection, and will ship strains for free with an order for their products).
- Include a phenol extraction step, followed by precipitation with NaAc/EtOH (Look for details in all the molecular cloning books). Otherwise you'll have problems like those you've just described (DNA disappearing upon incubation, etc).
- Add RNAse to the cell resuspension buffer you're using. This helps reducing (but will not eliminate) the amount of contaminating tRNA you are getting.
- Just in case, use freshly prepared alkaline lysis solution (it tends to acidify with time due to dissolved CO2).
You can add RNaseA to solution1 and store it at 4C, Solution2 should be stored at RT and prepared fresh every 2 weeks. If you have a disaperence of bands it means that one of the solutions contain DNase contamination. Prepere fresh ones and resuspend in the end instead in TE in DDW for 5 min at 37C.
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