plasmid extraction troubleshooting,please help !! - (Jun/02/2013 )
hi every one
i need help with plasmid extraction . my plasmid is PET21a and as u know it's an expression vector so it gives low concentrations in extraction ( or maybe i am the problem !! ) . any body has any suggestions to get higher yields ??
i truly appreciate ur help
We have no clue as to how you are doing it now. How could we tell you how to improve?
oh right ! i'm sorry
i do alkaline miniprep , u know the protocol , right ?
I know about 20 protocols, all of which are roughly categorized as "alkaline miniprep." Please tell us in detail what you are doing. How do you grow the cells? Culture medium? Antibiotics? What OD do you harvest at? What buffers are you using? What kit, if any? Columns? Did you add ethanol to the wash buffer?
All of these things make a difference, and could be the source of a problem.
here is how i do it ...
u know beside the low yield of plasmid concentration , another annoying problem i face is the huge amount of RNA even after Rnase treatment ! i would appreciate any kind of help with this problem too
BTW , it's transformed into DH5a !
I think you really mean 100 ug/milliliter ampicillin.
You don't specify the concentration of potassium acetate -- Adding it should produce a white precipitate. The supernatent should have a pH of 5-6 or so.
What is the pH of the phenol-chloroform. Commonly this is prepared at two different pH's: 7.5 for DNA extraction, and 4.5 for RNA extraction. Make sure you have the correct version. Read this page on phenol:
I would usually do a chloroform only extraction at this point to remove phenol, but the ethanol precipitation probably does this job.
I would add at least 2.5 volumes of ethanol, preferably a bit more. This is the most likely fault I see in your protocol. The spin should be at high speed, and you should note the expected location of the pellet. Be careful in removing the supernatent to avoid accidentally removing the pellet, both here and in the 70% ethanol wash. This is an easy place to lose your DNA. You could add pellet-paint NF (Novagen) to visualize the pellet the first few times you do this.
You should dry your sample after the 70% ethanol wash similarly to the previous way, and test by smell to see if the ethanol is gone. Five minutes of the tube open on the bench is usually sufficient. Do not over dry, which will make the DNA hard to dissolve.
You may want to dissolve in less than 50 ul, to increase concentration. Make sure to vortex the tube after adding the TE, so that the pellet dissolves.
You can add RNAse A to the initial resuspension buffer to help remove excess RNA. Also, note the pH of the phenol/chloroform is correct. Read this page on composition of the Qiagen buffers, especially P1, their version of GET: http://openwetware.org/wiki/Qiagen_Buffers
WOW ! u have A LOT of information !!
potassim acetate is 3M . i don't know the PH of phenol chlorophorm . i should probably try 2.5 volumes of ethanol this time .
hey would u answer some other questions please ?
1. what exactly does potassium acetate do ?
2. what about i use isopropanol instead of ethanol ?
1. The potassium acetate does two things. First, it neutralizes the NaOH in the lysis buffer, bringing the pH back toward neutral or acidic. Second, the potassium ions form potassium dodecylsulfate, which is more insoluble than sodium dodecylsulfate. This precipitates a white material, which helps to trap the genomic DNA during the high speed spin which follows.
2. Isopropanol works fine for the DNA precipitation, but is much less volatile than ethanol. You can use smaller amounts of isopropanol -- 0.6 to 0.8 volumes instead of 2.5 volumes. This may result in larger amounts of salt precipitating. I would continue to use a 70% ethanol wash, which will evaporate more quickly, and will be better at removing the salt.
there's another thing that the potassium acetate does.
the potassium ion, in the presence of ethanol (or isopropanol) will ionically bond to the phosphate in the dna backbone and precipitate the dna.
sodium, ammonium or lithium acetate can also be used to precipitate dna but in your case, as phage434 says, the potassium will also precipitate the sds.