can someone tell me about extraction DNA from polyacrylamide gel? - (Nov/16/2006 )
Hey everybody, I'm very glad to join this forum. I'm a final year student and I have to do some experiments with SSR (simple sequence repeat) indicator. Now I'm preparing to cloning some genes and I have some troubles. It's the DNA extraction stage, but from 6% silver- staining polyacrylamide gel (normally, in my lab, they just extract from agarose gel). I need some advices about the protocol and the difference between the ethidium bromide - staining polyacrylamide gel and the silver - staining one which I have to noticed before I go to extract DNA.
The interested band is about 300 bp and I have to electrophoresis PCR products on polyacrylamide gel because there're many bands which have their size near the band I need (e.g 350bp, 270bp..). Can I use the QIAEX II Gel extraction Kit of QIAgen for this purpose? Other than this Kit, is there any procedure I can use? (this Kit is too expensive, so if there's any cheaper way, it's better)
Please help me, thank for your help!!!
if your DNA band were weak and contains less amount of DNA, whcih can only detected by silver stained band on PAGE, you might cut the band and put it into boiled water for elution, then reamplied for purification,
if your band is strong and can be detected by EB which is less sensitive than silver staining. you might followed instruction of QiaexII kit.
have you ever boiled polyacrylamide gel slice in water, I don't believe that the recovery DNA is enough to do the reamplification because in hot water, most of material in these slices will be denatured.
How can you stain gel with EB, or it's the same way with agarose gel?
check how people recovery DD-RT band from PAGE gel , they even boil the gel slice for 15mins, I just put boiled wate into tube with cutted band...
stain PAGE with EB is the same as agarose gel, put gel in EB solution (it is better using the same buffer for electorporesis)
by the way SBRY green is more senstive for staining of DNA in PAGE gel.
, thank you so much for your answers! But my gel size is about 33 X 35 cm, so it's too difficult and unsafe when trying to stain it in EB solution. Do you know about another procedure which runs the gel slice in 2% agarose and obtain DNA on the DEAE membrane which is embeded below wells? i found it when I'm searching more information but I don't know how I can buy this membrane.
mini size PAGE should work for such size products (such as gel used on BioRad mini II)
alternatively low melting temperature agrose might be considered
I thought about it before and when i asked my teacher, she said that the mini size PAGE isn't appropriated because the length of it isn't enough to discriminate between this band and some another bands approach to it. I implied the electrophoresis apparatus which is used for protein (about 10 X 13 cm ?), it would be easier for me to work with it
10% non denature PAGE with better resolution for 300bp DNA around
You're a kind man! I will think more about all your advices. Hopefully I will successful with my experiments but I think that there will be more troubles I have to face, so I will post all of unclear things with me and glad to receive more help!
cool! I mean photo