6 replies to this topic

[mahsa]

hey guys
I gotta extract a DNA fragment from a double digestion reaction. the problem is the size, the fragment is only 80 bp. Is there any better solution than running it on a agarose gel and then using Phenol/chloroform extraction prior to ethanol precipitation?
really appreciate any idea.
await anybody's reply in anticipation.

[pDNA]

not really ...but it should be no problem doing it the way you describe ...use a higher percentage agarose gel (1.5%).

Alternatively, you can try the Qiagen QIAEX II System ...works really fine for the recovery of small fragments!

Regards,
p

[perneseblue]

you can use 3% agarose to gel purify the DNA fragment. But you will probably get better resolution with PAGE.

What do you want to do with this tinny fragment?

[Sagesigh]

So we make an instrument that makes extractions like that automatically. We are working on a less than 100bp cassette now. The instrument, Pippin Prep, is currently being sold to sequencing labs for nextgen sample prep, but it is really a general purpose gel purification tool. Our hope is that by making gel purification ridiculously easy (mostly just pipetting in your samples and pipetting out the desired band or size range from each after the run), that people will use it for a wide range of applications.

Yields also tend to be high, usually 80% to 95%.

If you are curious, see sagescience.com

[mahsa]

pDNA, on Jun 9 2010, 11:48 AM, said:

not really ...but it should be no problem doing it the way you describe ...use a higher percentage agarose gel (1.5%).

Alternatively, you can try the Qiagen QIAEX II System ...works really fine for the recovery of small fragments!

Regards,
p

tnx a lot P
i have already seen the QIAEX II System kit but the problem is i have to do the job as cheap as possible, so i think am gonna have to go for the phenol/chloroform + ethanol precipitation for now.

tnx a lot again
all the best mahsa

[mahsa]

perneseblue, on Jun 9 2010, 09:05 PM, said:

you can use 3% agarose to gel purify the DNA fragment. But you will probably get better resolution with PAGE.

What do you want to do with this tinny fragment?

well, i have already run the 3% agarose gel and the band is relatively sharp and seems extractable. i have to get the tiny DNA out. it is going to be cloned in another plasmid and hopefully be expressed in mammalian cells. the problem is i have no money to buy a new extraction kit with appropriate cut off so am gonna have to go with traditional methods.
thank you
all the best
Mahsa

[mahsa]

Sagesigh, on Jun 10 2010, 07:55 PM, said:

So we make an instrument that makes extractions like that automatically. We are working on a less than 100bp cassette now. The instrument, Pippin Prep, is currently being sold to sequencing labs for nextgen sample prep, but it is really a general purpose gel purification tool. Our hope is that by making gel purification ridiculously easy (mostly just pipetting in your samples and pipetting out the desired band or size range from each after the run), that people will use it for a wide range of applications.

Yields also tend to be high, usually 80% to 95%.

If you are curious, see sagescience.com

Thank you very much indeed for the help, I can't afford a new kit right now, but the site is informative and helpful.
Mahsa