Hi Anand....

The original protocol used first step of Phenol:Chloroform (25:24) and subsequent step of chloroform:isoamyl alcohol (24:1)......But now-a-days many labs use Phenol:Chloroform:Isoamyl alcohol (25:24:1) in the first step followed by the same second step....You can go either way..some claim adding Isoamyl alcohol right in the begining is advantageous..

Dear Dr. Mohandas

You can use the standard procedures for nucleic acid purifications ..You can purify the PCR product using Phenol:chloroform, followed by chloroform:isoamyl alcohol and precipitate by a variety of methods (sodium acetate and ethanol/isopropyl alcohol etc)...you can find the procedural details in any lab manual..

Even in this era when we have kits for almost everything..these are very widely used for nucleic acid purification and work perfectly...

@ Ajaya.. Just I want to clarify the doubt we-their, we need to use Phenol:Chloroform:Isoamyl alcohol (25:24:1) ratio or just we need to use Phenol:Chloroform (25:24) for purification......

Hi Anand....

The original protocol used first step of Phenol:Chloroform (25:24) and subsequent step of chloroform:isoamyl alcohol (24:1)......But now-a-days many labs use Phenol:Chloroform:Isoamyl alcohol (25:24:1) in the first step followed by the same second step....You can go either way..some claim adding Isoamyl alcohol right in the begining is advantageous..

Thanks Ajay for your guidance.

I used in the past esterilized glass wood in order to eliminated the agarose and afterwards ethanol precipitation at acid pH

If you have a magnet, you can use magnetic beads to clean up the DNA. This is both extremely cheap and non-toxic:

Add 2 µl Seradyn magnetic beads (Thermo Cat# 4415-2105-050250), and 20% PEG8000/2.5 M NaCl to give 9.6% PEG8000/1.2 M NaCl final (e.g. to a 50 µl PCR reaction, add 48 µl of the PEG/NaCl solution), mix well, let stand for 5 min at room temperature. DNA > 100 bp adsorbs to the beads in the presence of PEG/NaCl, leaving the primers in solution.

Collect the beads with the magnet, wash them twice with 200 µl 75% EtOH (this can be done simply by applying the magnet several times on opposite sides of the tube, causing the beads to zip through the EtOH).

Air-dry and elute DNA off the beads in the desired volume with TE.

Ligations work even in the presence of these beads, and the ligation product can be desalted (e.g. for electroporation) by simply adding PEG/NaCl again, and following the above procedure.

You can always run the digested product in low melting point agarose and perform an in-gel ligation.

There are also protocols for making homemade glassmilk which you can use to extract your DNA from agarose and elute. Here's a reference: BOOM, R.; SOL, C.J.A.; SALIMANS, M.M.M.; JANSEN, C.L.; WERTHEIM-VAN DILLEN, P.M.E. and NOORDAA, J.V.D. Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology, March 1990, vol. 28, no. 3, p. 495-503.

Hi! Can I use any of these techniques of purifying PCR product to improve RFLP results. I do not get any band after ALU-1 or Hha-1 enzyme treatment since my PCR product has less DNA concentration. I tried to increase the yield of PCR product by using higher concentration (4 ul instead of 2 ul) of DNA in primary PCR and 2ul of the product in nested PCR instead of 1 ul. This did not give me more product. Can anybody suggest a solution.

Hi Chhaya

I hope my suggestion is still not very late.....if by any chance you cannot increase the yield of the PCR product and you still want to to carry out restriction analysis......I'll suggest two things

1) Do PCR in multiple tubes..pool them, purify and precitate the products and dissolve in less volume for downstream analysis.

2) alternatively...you can analyze the results of restriction analysis on a more sensitive platform..such as PAGE gel followed by silver stainin

bye

Isoamyl helps define layers so easier to separate during extraction