If I want to extract large plasmids from bacteria (more than 100 kb), what is the best way and best kit to get them in pure and high yield.
Please help me urgently if you can
Best Regards
Nucleobond has a great BAC purification kit - check them out. I used it to extract and inject 150kb transgenes into mouse embryos and was happy with it.
Nucleobond has a great BAC purification kit - check them out. I used it to extract and inject 150kb transgenes into mouse embryos and was happy with it.
From my experience, I will suggest for purification of the large amount and ultrapure plasmid DNA, QIAGEN Plasmid Mega Kit. The expected yields are ranging 0.5–2.5 mg depending on low or high copy plasmids
Hi, plasmid extraction kits, sure are efficient and useful but if you are looking for a rapid, easy and inexpensive method, please find attached a very good protocol that could extract plasmids of up to 200 Kb.
i too suggest using QIAGEN Plasmid Kits for Plasmid Extraction for high yields.
http://www.qiagen.com/in/products/catalog/sample-technologies/dna-sample-technologies/plasmid-dna/
Most plasmid kits cannot cope well with larger plasmids as they are designed around cloning vectors. Plasmids of around 60-80kb are usually extractable (such as Salmonella virulence plasmids). Large plasmids 100-250Kb usually have lower copy numbers and given their size are hard to extract. Older methods such as ultracentifugation and phenol:chloroform extraction may work. I would seriously consider BAC-based methodology and wish this was around when I was trying to extract 220kb plasmids in the 1990s.
Grow 5ml over night culture. spin down, folllow qiagen protocol, but adjust to 300 ul p1, 300 ul p2, and 420 p3. Elute by adding 50 ul H2O, incubate in 70C for 30 min. Spin down. You will got what you need. Miniprep big plasmids, I always do this way. Yield increase 50 times.
High Pure Plasmid Isolation Kit(250 preps) BRAND-Roche-Cat.No- 11754785001
that is best
NucleoBond by MN is also an option. in case if qiagen doesnt work and if you can get a sample kit of this product, u can try this one also.
Hi there.
QIAGEN by far should be your choice. You get much more material back and of higher purity. I personally do not like MN kits at all, the are cheaper but not as effective.
Best wishes,
Nikos
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