Looking for midi and maxi plasmid isolation protocols from E coli (high copy number plasmid) cultures above 100ml (LB)
Protocols tried so far:
Sambrook's midi and maxi prep
Alkaline lysis method
Boiling-Lysozyme-IPA-EtoH method
PEG mediated plasmid isolation procedures.
Issues consistently facing:
Not able to completely avoid Genomic DNA during bulk plasmid isolations (Though plasmid is present in eluted sample).
Sometimes experiencing RNAse contamination (even after RNase Treatment what about using Lithium Chloride to get rid of RNA during lysis step and neutralization step). Are there any standard ratio for pellet weight to RNAase concentration to be used
Precautions taken/modifications:
Considering the lysis step being important in denaturation of genomic dna and plasmid dna, we did try giving very less incubation time ~1min for lysis step and compared it with 5 min incubation with lysis. In both cases only the amount of genomic DNA was high in 5 min incubation. and for both lysis and neutralization steps care was taken to invert mix gently with out giving any chance for shearing Chromosomal DNA.
Apart from using any commercial kit methods for midi and maxi, are there any inhouse developed or in practice protocols please share links/procedures
Downstream application of bulk plasmid:
Restriction digestion alone.
this link is useful
http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NuBoXtra.pdf
regards
Hello Hassan , Thanks for the link, but i am exactly looking for non column, non commercial kits based conventional midi or maxi prep inhouse developed protocols
follow these links
https://www.researchgate.net/deref/http%3A%2F%2Fwww.mn-net.com%2Ftabid%2F10598%2Fdefault.aspx
https://www.researchgate.net/topic/Maxi-Prep
Forget about the DIY kit option! Send us your plasmids as we can prep them for only a fraction of the price offer by other companies. You actually save money if we do it for you! By using our own proprietary protocols, we can not only offer the best price to our customers but also obtain better results than you would with a kit in terms of DNA quantity and quality. See for yourself at https://www.biozilla.bio/plasmid-dna-purification
- Badges
- Science topic