I am trying to clone few genes within a very low copy no (7.4Kb) plasmid. The yield is pretty low after Mimiprep from 5ml of over night culture, eluted in 30 microliter of water/TE buffer. And after digestion purification I am loosing most of it. What I need to do?
If the yield is low after plasmid extraction, then of course you wont get much after digestion. What you can do is to increase the plasmid yield:
1) if you have a lot of tubes of harvested plasmid, you can pool all the tube together and do ethanol or isopropanol precipitation to get a higher concentrated plasmid.
2) if you do not have multiple tubes of harvested plasmid, you can re culture and extract the plasmid again. Let's say you harvest one tube of plasmid from 5ml of culture, in this case, you culture multiple 5ml culture. Harvest them as usual but combine them when you put into column. Let's say you are harvesting 4 tubes, after lysis and neutralization, you centrifuge it as usual, but later you combine all supernatant from 4 tubes into one column so that you can harvest higher amount of plasmid per tube. Then only you proceed with digestion.
Hope this helps!
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