low concentartion of DNA after miniprep - (May/27/2008 )
hello there
i know that usually DNA yield from miniprep is low but to increase it we usually do phenol ,chloroform/ethanol protocol but this time i did all that and still got DNA concentration as 0.178 ???? what is the problem or factor affecting mimniprep?
thankx
help any ideas?
what kind of miniprep are you doing? If you are using a kit it often helps to warm up the elution buffer first. It's also possible that you have a low copy plasmid, in which case you might want to consider doing a midiprep instead.
thankx for your reply
i am using promega miniprep kit
my plasmid is pcDNA3.1+ , its a high copy plasmid
i am thinking that i am loosing my DNA at some step but i dont know which one?
thankx anyway
I would guess that the DNA is being lost at either/both the initial binding to the silicon column and the elution from said column. DNA is usually lost at these two steps.
To improve binding, you can add isopropanol (3 volumes solution:1 volume isopropanol). I hear adding (1:10 volume) 3M NaAcetate helps, but I have never tried.
Also pass the eluted solution through the column several times (3x) gives the DNA more chance to bind.
To improve elution, well there is using warm elution buffer.
When dealing with column, I divide my elution buffer volume in 40/60. (Say I want a final volume 50ul). I first elute with 20ul. Then I elute the column again with 30ul. I also leave the elution buffer in the column for about a minute.
As for plasmid/bacteria cultivation, well you can use a richer medium (eg SOC) or larger culture volume. If the plasmid is suspected to be unstable, reducing the cultivation temperature helps (as low as 28 Celsius).
Thank you soo much
it was really helpfull
it was really helpfull
So now what's the yield?
Have you done it yet???
If you want good yield , there's always the old school method of you know the original miniprep ( no spin column involved)
Lysozyem -> lyse+ NaOH --> Neutralize->phenol/chloroform-->etoh precipitate.