Removing PCR primers

kim at m44.unm.edu kim at m44.unm.edu
Wed Jun 3 10:38:15 EST 1992


In article <1992May29.072159.19202 at bronze.ucs.indiana.edu>, jgraham at bronze.ucs.indiana.edu (the End) writes:
>
>Greetings,
>
>We now generate our templates for in vitro transcription by PCR
>instead of isolating restriction fragments. I have read a report of
>using Sepharose CL6B column chromatography to remove the remaining
>primers from the reaction product. However I would prefer a spin
>column procedure. Has anyone tried using spin columns made with this
>or any other resin to remove primers of about 20 nt from DNA fragments
>around 500 bp ? 

I have been making antisense RNA probes using the Stratagene in vitro
transcription kit with PCR products as the template.  This is, roughly, the
somewhat dirty procedure I use:

Prepare the PCR reaction mixture on ice.  (I use a 20 microliter volume)
Dip a yellow tip into a colony of rescued pBluescript clone and place the tip
  into the PCR reaction mix.
Twirl the tip a little, and pull it out.  Add oil, and run 30 cycles.
Add proteinase K to 50 micrograms/ml and incubate 37 C for 30 minutes.
Phenol extract twice
Chloroform extract once.
Ethanol precipitate the PCR product.
Spin out the DNA, wash with 70 % ethanol and dry.
Make up a 5 microliter in vitro transcription mix (I don't need very much
  probe for my work). on ice.
Use the transcription mix to dissolve the PCR product, then incubate 37 C for
  30 minutes.
Kill the reaction and run it through a spin column to remove unincorporated
  NTP's.  You now have probe.

I have only been able to get about 50% incorporation, which is about 4 - 6 x
10^6 cpm in a reaction, but this is sufficient for my use right now.

I hope this is of some use.

Daniel Kim



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