I was trying to clone a gene into EcoRI site of pUC-18 by shotgun cloning method. A fragment of approx. 15 kb gets cloned and now I am trying to isolate the recombinant plasmid. However, every time I get DNA bands with the smear on agarose gel. I checked everything for external nuclease contamination and I am sure there was no nuclease contamination during the procedure of isolation of recombinant plasmid. Please suggest any modified methods of alkaline lysis of Sambrook or any other methods for isolation of this plasmid, so that I would able to get a recombinant plasmid without any smear and with the high yield.
we normally use one step mini-prep method using phenol, chloroform, isoamyl alcohol. that works for high molecular weight....
Nucl. Acids Res. (1991) 19 (10): 2792.
If you don't want to use phenol you could use the Xtra Midi kits from Macherey-Nagel which are capable of isolation large constructs up to bacmids. If I remember rightly, the JM-strains contain high levels of nucleases and you have to wash the bound DNA thoroughly.
I would try a miniprep kit. Qiagen miniprep kit comes with an additional clean up buffer (PB). Zymo Research often has test samples for their kits that you can request for free. For miniprep kit: http://www.zymoresearch.com/dna-purification/plasmid-dna-purification/zyppy-plasmid-miniprep-kit. On the right side, there is a link for a free sample.
You should be able to treat them as any BAC (bacterial artificial chromosome). As noted above, Qiagen makes some good kits (see attached).
Qiagen miniprep kit. Process your culture (e.g. 15 ml) in 5 ml aliquots and reuse column. Incubate 30 min in fridge after neutralisation step. Worked for cosmids (40 kb) perfectly.
I'm surprised that you are having trouble with plasmids this small. Usually problems don't start until you get above 40 kb. You might like to try the original Birnboim-Doly method. My experience is that this works well up to 50 kb. It's also cheap and simple. Just make sure that your sodium hydroxide solution is fresh. Avoid vigorous mixing (like vortexing); this might shear your plasmid.
Supplement: Watch out for kits that are based on anion-exchange resin, because they normaly provide isolated DNA of higher quality compared to mini kits that are based on silica membranes.
- Badges
- Science topic