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DIY PCR Cleanup/Gel extraction and miniprep solutions

PCR Cleanup gel extraction miniprep recipe

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#1 Bio-Lad

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Posted 01 October 2014 - 07:58 PM

   Since we can recondition silica columns in order to reuse them, I've been making my own solutions for Minipreps, PCR Cleanup and Solubilization/Extraction of bands from gels.  Below, I've included the recipes to make these which, in addition to the reconditioned silica columns, have saved our lab from having to buy any of these kits over the past 4-5 years (and I go through a LOT of these).  Hopefully this will be useful to others.

 

Membrane Binding/Gel Solubilization Solution

(4.5M Guanidine Isothiocyanate, 0.5M KOAC pH 5)

Steps

For 50mL

For 100mL

1) Add Guanidine Isothiocyanate (GIT) to 1M KOAC, pH 5.0 in a beaker.

26.6g GIT

+

25mL KOAC

53.17g GIT

+

50mL KOAC

2) Place on a warm hotplate with constant stirring until the guanidine is mostly dissolved.  A setting of 2 on the hotplate works fine.

3) Pour contents into a graduated cylinder and QC to the final volume with MilliQ water.  This should not require much additional volume.  Put the beaker back on the stir plate and continue stirring until everything is in solution.

4) Filter into a container using a 0.45µm filter.  There is no need to adjust the pH.  The solution is ready to be used at this point.

 

  • To use for PCR cleanup, add an equal volume solution to PCR reaction and mix.  Add to a silica column and process as usual.
  • To use for bands cut from gels, add 1mL solution per gram of gel slice and incubate at 55C for 10 minutes with occasional mixing.   Add to a silica column and process as usual.

Miniprep Solutions

for use with these columns (there are variations on these.  This is what has always worked well for me)

 

Cell Resuspension Solution

 

Final solution: 50mM Glucose, 10mM EDTA, 25mM Tris HCl pH 8.0

Optional, add 1mL of 10mg/mL RNAseA/100mL for a final concentration of 100µg/mL (I use without and it works fine).

 

Cell Alkaline Lysis Solution

Cell Lysis Solution

0.2M NaOH (8g/100mL)

1% SDS (1mL 10% SDS)

QS to 100mL

 

Neutralizing Solution

 

 

Reagent

Final Molarity

Amount/250mL

guanidine hydrochloride

4.09M

97.7g/250mL

potassium acetate

0.759M

18.625g/250mL

glacial acetic acid

2.12M

30.5mL/250mL

 

  1. Add the three reagents in a glass beaker and then add MilliQ water to ~90% of the final volume.
  2. Mix the solution using a stir bar and stir/hot plate to mix.
    1. Setting the stir plate to a heat setting of 2 while mixing helps speed up the endothermic mixing process.
  3. Once the majority of the reagents have entered solution (a small amount will remain out), pour the solution into a graduated cylinder and add MilliQ until you have the final volume.
  4. Pour back into the beaker and stir if needed.  At this point the solution should be completely clear.

Obtain a pH.  It should be around 4 (I get ~3.85)

 

Miniprep/Silica-Based DNA Purification Column Wash

 

This is the solution used by Promega and works with silica-based column purifications including minipreps, maxi preps, and PCR/Gel cleanup kits.

 

Final Solution: 60% EtOH, 60mM K-Acetate, 8.3mM Tris-HCl, 0.04mM EDTA.

Recipe (for 1 Liter)

         600mL 100% EtOH

         5.9g Potassium Acetate

         8.3mL 1M Tris pH 7.5 (Lab Stock)

         80µL 0.5M EDTA pH 8.0 (Lab Stock)

         QS to 1L with MilliQ water

 

 

For PCR cleanups & gel extractions, I use the above solutions and follow the protocol for the Promega Wizard SV Gel and PCR Clean-Up System.

For minipreps, I use the above solutions and follow the protocol for the Promega Wizard SV Miniprep kits.


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#2 bob1

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Posted 04 October 2014 - 08:51 PM

Nice. You could try posting these to openwetware too.

#3 Bio-Lad

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Posted 05 October 2014 - 04:32 AM

Good idea! I might just do that.

Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/






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