I wanted to purify and isolate my DNA from agarose gel, so I wish to know what type of gel and what percentage of agarose I can add to make the gel and how to isolate DNA after the run. Apart from using column purification method.
Another fun way to get your band out (my personal favorite). Run gel, check position of band of interest. Now cut a 'well' in the agarose directly below the band. Then run the band into the new well and pipette it out.
Two words of caution. 1) If the level of the running buffer is higher than the top of the agarose slab your band will diffuse away. 2) if you stained the gel with ethidium bromide it will still be stuck to your DNA. I usually locate the band by UV shadow without stain and adjust the running buffer level after I cut the new 'well' so that there is little or none on top of the gel.
The percentage of gel you will need to run depends on the size of your DNA fragments, I would also run the gel slower and at a lower voltage to ensure good band separation - TBE buffer if you have it.
Would electroelution of the DNA be possible?
http://rothlab.ucdavis.edu/protocols/dna-electroelution-2.html
Good luck :)
just use Qiagen kit for Gel extraction, you need to cut the DNA band with a piece of gel around it transfer it to eppendorf tube , weight it then follow the kit instruction it is so easy
good luck
I second Taha's suggestion. It's very easy to cut out the band, and purify with a suitable kit. Wizard SV Gel and PCR Cleanup Kit from Promega works fine, as well.
The quiagen kit works reaaly good and gives a great clean fragment.
An alternative to the kit-column methods is to use low melting point agarose for the gel, cut the band out and then melt it at 65-70C and phenol extract it. Just Google
"phenol low-melt gel extraction" to get a couple of protocols. Of course, if you don't have low-melt agarose and phenol on hand, I would buy one of the kits already mentioned, as they are much easier and quicker with more reliable yields. It's also important to remember to avoid exposing your gel to short wave UV light at any time. Use a transilluminator or hand-held lamp with long wave UV to visualize your band and to cut it out. Just a short exposure to short wave UV can severely damage the DNA for use in subsequent steps.
Hello..You can use comertially available kit to extract your DNA from the gel. Ambion/Qiagen kit for Gel extraction, you need to cut the DNA band with a piece of gel around it transfer it to DNA ase /RNAase free eppendorf tube , follow the instructions in the kit ..as per their recommendations..Its quick method and it is so easy to get your interest of DNA/
Another fun way to get your band out (my personal favorite). Run gel, check position of band of interest. Now cut a 'well' in the agarose directly below the band. Then run the band into the new well and pipette it out.
Two words of caution. 1) If the level of the running buffer is higher than the top of the agarose slab your band will diffuse away. 2) if you stained the gel with ethidium bromide it will still be stuck to your DNA. I usually locate the band by UV shadow without stain and adjust the running buffer level after I cut the new 'well' so that there is little or none on top of the gel.
First choose the percentage of gel according to your PCR product size. If you possible to get Zymoclean™ Gel DNA Recovery Kit, you can elute the good quality DNA.
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