Looking for some suggestions, for a colleague!
She is working on Minicircle DNA technology. The parental plasmid has the size of 5237 bp and the Minicircle is of size 3495 bp. She is using ZYCY10P3S2T modified E.coli strain. While doing mini-prep, the genomic DNA contamination is near to Nil. But while doing Midi-prep, she gets a lot of genomic DNA contamination. I suggested chilling and pre-warming of buffers and pre-warming of elution buffer, gentle mixing of P2 buffer, 10 h overnight culture instead of 18 h, and use of 25 ml of culture volume for Midi-prep. She has done pH adjustment also. She has also done linearization of parental plasmid using REs not present on Minicircle and she has used Minicircle safe DNase for digesting parental and genomic DNA. Yet, she keeps getting genomic DNA contamination.
She is using Thermo Scientific kit.
Are there any other suggestions, that are worth trying?
Picture Legend:
The first two lanes are controls - the minicircles and the parental plasmids extracted by miniprep - they show no larger- sized molecules - so genomic DNA is almost NIL
But the rest of the lanes - in which the lanes next to the controls are minipreps again - have the parental plasmid (I didn't do induction) - but also the genomic DNA, the size mucb higher than the ladder
The last two lanes are extracted by Midiprep
They also contain a higher volume of genomic DNA.
Very interesting to see this result. Never seen yellow bands before. Anyway, parking here for future answers✨👀❤🌃✨
The yellow is an artifact of the imaging system (mine highlights the saturated pixels in red).
Ok, on the main question at hand. The large size bands near the wells do look like gDNA, and possibly some other material carryover as well, they are quite smeary.
A few suggestions:
use less starting material, sometimes it is worth it to have a lower yield but a cleaner product.
try to avoid carryover of any debris from the cleared solution after lysis, while the cleanup steps do help remove contaminants, having less cell debris to start with will help a lot
If this is the resin column kit, be sure to let all solutions drip through (don't use pressure or a vacuum to speed it up)
if this is the membrane kit, make sure the membrane isn't getting damaged or you can get flow through of larger materials.
Hope this helps!
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