How to purify and clone a 88 bp fragment? - (Oct/03/2005 )
I am trying to insert a 88 bp framgemt into a plasmid. I was wondering how to purify such a small fragment since most kits have >100bp as a size limitation. I have tried anyway with some kits but I lost almost all product.
where is this fragment from?
i've read once an extraction protocol :
it was from Jeff Lawrence
1. Remove gel slice contain DNA fragment and place in 10 volumes of:
300 mM NaOAc, pH 7.0 (300 ml 1 M NaOAC, pH 7.0)
1 mM EDTA (2 ml 500 mM EDTA, pH 8.0)
698 ml ddH20
2. Incubate at 22 C for 30 min. Transfer gel slice to a fresh tube.
3. Place tube in a Dry Ice/Ethanol bath for 5 min.
4. Puncture the bottom of a 0.5 mL microcentrifuge tube with a needle. Place the gel slice into this tube. Place this tube inside a 1.5 mL microcentrifuge tube.
5. Centrifuge for 15 min.
6. Collect the Eluent from the 1.5 mL eppendorf tube. Extract and precipitate the DNA.
maybe you can try. The dna remains in te eluent an may be precipitated by salt/ethanol method.
Small products can diffuse quite happily from agarose blocks. We used to cut small bands (up tp 300 bp) from gels and place in a tube with a small amount of dH2O (just enough to cover the block). We'd leave it overnight for the DNA to diffuse out and then spin the tube in a centrifuge at max speed. You could then aspirate some of the solution into a new tube. We would use the DNA in PCR and it would always work...however, I am not sure if there would be enough DNA for a ligation but its something to think about.
Thanks! I will certainly consider your suggestions!
A bit similar to the above mentionned techniques I use the simple migration to a whatmann paper
you make an incision into the gel below the band with a blade and insert a piece of whatmann paper just between both agarose part.
you run again slowly until the band has migrated onto the paper (you can chek under the UV lamp).
You remove the paper with forceps and place it in a 1.5ml eppendorf tube that you previously pinched a hole with a needle (1.2 pink needle works great, Be careful with your finger)
Spin down the tube on top of another 1.5ml tube and collect the liquid coming out of the paper. Treat with Phenol chloroform and EtOH precipitates and that's it it works fine with any size of DNA.