5 replies to this topic

[hotstuffdb22]

Hello!

I really need some help!  My final goal is to make high quality DNA that can be labeled and used as a probe for southern blot.  To this end, I have been isolating PCR products from an agarose gel, followed by Qiagen's gel extraction kit.  However, my DNA doesn't seem to be that good, as there is often a hard time in using this DNA to make a radioactive probe for southern blot.  Thus, I thought an alternative way to make probe DNA would be to do the same PCR reaction, but do a PCR purification instead of a gel extraction.  Are there any advantages or disadvantages to this approach?

[phage434]

If you have a single clean band, there is no problem with using PCR purification instead of running gels. My current favorite PCR purification is to use Ampure beads and magnetic separation, which is faster, higher throughput, and avoids all column handling. Also robot friendly.

[hotstuffdb22]

Thanks a lot for the advice.  I'm not familiar with the Ampure beads and magnetic separation.  Can you post a link to a website so I can purchase?

[phage434]

Ampure beads may be unreasonably expensive to purchase in small (or large!) quantitites, but they are very convenient. Beads + multichannel pipettor + 70% ethanol and a magnet plate will work far faster than columns for more than, say, four samples. Here's a protocol for making your own "Ampure" beads more cheaply. You can also size-select DNA fragments by controlling the PEG concentration of the final bead + sample mix. High throughput NGS sequencing sometimes uses this for size selection of sequencing fragments.

 

I found that you should use less Tween-20 than indicated in this protocol.

Attached Files

•  serapure_v2-2.pdf   228.14KB   372 downloads

[phage434]

This is the plate we use for separation. It's on sale until 20 December. They also make a low elution (LE) plate for elution volumes of 10 ul rather than 40 ul in the standard plate. I have no experience with that plate yet. I mostly use these with 8 well PCR strips when doing things manually.

 

http://www.alpaqua.c...spx#46630-order

[bob1]

You can also incorporate your labeled nucleotide into the PCR that is producing the probe (I recommend DIG labels for Southern blotting), so that there is no need to add the label after amplification.

 

I also second Phage's advice about using beads or other PCR purification rather than gel extraction.