DIY PCR Cleanup/Gel extraction and miniprep solutions - (Oct/01/2014 )
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)
1) Add Guanidine Isothiocyanate (GIT) to 1M KOAC, pH 5.0 in a beaker.
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.
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
glacial acetic acid
- Add the three reagents in a glass beaker and then add MilliQ water to ~90% of the final volume.
- Mix the solution using a stir bar and stir/hot plate to mix.
- Setting the stir plate to a heat setting of 2 while mixing helps speed up the endothermic mixing process.
- 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.
- 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.
Nice. You could try posting these to openwetware too.
Good idea! I might just do that.