Now we are working on extracting large-sized plasmids(55kbp,70kbp,100kbp) from E.coli EPI300 by traditional DNA ethanol precipitation method without the spin column. But we can't get good results (Gel electrophoresis does not show distinct, singular target bands.) PS: The colony PCR validation results indicated that the plasmid has successfully been transferred into EPI300.
Any suggestion is welcome. (any commercial kit, methods, protocols)
Thanks.
Continuous method for large-scale extraction of plasmid DNA by modified boiling lysis method: This protocol describes a streamlined method of plasmid DNA extraction by continual thermal lysis, which can be suitable for processing large volumes of E. coli cultures.
Remember that the positive result of the PCR colony does not mean the complete confirmation of the presence of the gene in your plasmid. To be sure of the true or false positive result, please repeat the PCR test on the bacterial colony without plasmid and on the plasmid without the target gene as a control.
Look dear the same thing happened to me, i tried to extract the plasmids with different molecular weights by manual procedures, but no results were gained so finally I used the kit provided by Qiagen and I gained an effective extraction
Normal mini preps protocols will probably not be effective to get large plasmids in intact form. You need a gentle lysis procedure and I would stay away from denaturation steps such as the traditional 3 solution mini prep procedure. These large plasmids behave much more like chromosomes than they do like plasmids biochemically. I would look for protocols or kits designed for large plasmids, these would especially have been prevalent in the era of cosmids and BACs which are in a similar size range.
Hi, for bacmid extraction we proceed as with small plasmid (3ml of preculture/pellet/solution sds NaOH/then acetate de K...phenol/chloroform/EtoH precipitation) exept that we did not vortex (just invert tubes up and down by hand 5-times at each step). However the yield is very small as large plasmids are not in multiple copies in the bacteria (so it is not easy to see them on a gel; they were only used to transfect SF9 cells) If you want to do restriction map for example you will need to start with larger volume. there is kit to extract bacmid (Qiagen, Thermo-fischer....)
Hi, other solutions:
One method that is commonly used for isolating large plasmids is the cesium chloride (CsCl) gradient ultracentrifugation method. This method allows for the separation of DNA based on density, and it can be particularly useful for isolating large DNA molecules such as plasmids.
Another approach is to use specialized plasmid extraction kits that are designed to handle large plasmids. These kits often include modified protocols and reagents that are optimized for the extraction of high-molecular-weight DNA.
One popular kit for isolating large plasmids is the "QIAGEN Large-Construct Kit," which is designed for the purification of large plasmids and BAC DNA. This kit is suitable for isolating plasmids up to 100 kb in size and provides high yields of pure DNA.
Another option is the "NucleoBond Xtra Midi EF" kit, which is designed for the purification of ultrapure endotoxin-free large DNA, including BAC and PAC DNA. This kit enables the isolation of large plasmids up to 200 kb in size.
Both of these kits are optimized for isolating large plasmids and can provide improved yields and purity compared to traditional methods.
- Badges
- Science method