Hi all,
I am working with a pME6032 shuttle vector with cloned genes (approx 10-11kb in total) transformed to E.coli DH5-alpha.
After alkaline-lysis method to obtain the plasmid DNA pellet. I then proceed to PEG precipitation in the following manner:
1. resuspend pellet in 1ml 1x TE buffer
2. add 1ml of 13% PEG in 1.6M NaCL, vortex briefly, incubate on ice for about an hour
3. Spin 15000rpm, 5mins
3. Phenol-chloroform purification 2x
4. Chloroform purification once
5. Add 1/10 volume sodium acetate and 1ml 100% isopropanol (2ml total vol)
6. spin 15,000rpm, 5 mins, discard supernatant
7. wash with 70% ethanol, spin 15,000rom 2 mins, discard supernatant
8. dry pellet, resuspend to a total volume of 100 microliters.
So the problem is that, even though I can obtain plasmids, the yield is too low for a large prep. average yield using this protocol is 0.3-0.8mg/ml whereas previously when I followed the same procedure on a pRSETB (3kb plasmid), I can get as high as 5-7mg/ml concentration.
Im thinking if the problem is in the PEG concentration or the length of the precipitation in PEG (I've read in some protocol that they incubate the plasmids overnight).
So any feedback will be greatly appreciated.
Thank you very much.
Dear Kris,
You see this difference because pRSET B harbors pUC origin of replication that yields ~300-500 copies per bacteria; pME6032 on the other hand contains p15A origin of replication that yields ~10 copies per bacteria. Nothing wrong with your experiments, it is just the copy number difference between the two plasmid vectors. One more thing, the p15A -based plasmids can be amplified (copy number can be increased) by using spectinomycin.
I was also thinking about the copy number but do not know how to explain it. Thank you very much for your kind feedback and insights! this is truly helpful.
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