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I expressed a yeast vector in E.coli and purified about 13µg of it. I then linearized it using a restriction enzyme, and attempted to gel purify it. I attempted this twice. The gel showed a clear bright band at the right spot, but after gel purification I end up with only 0.7µg of linear plasmid. I doubt I made the same mistake twice. What is the best way to purify a linear plasmid after digest?

Edit:

The plasmid is about 3.5 kb. I incubate an overnight culture of E. coli, miniprep the following morning, elute into water. I end up with 13µg of DNA in 200µl of H2O. I add the PmeI (10µl) and the CutSmart buffer (23µl) from NEB, incubate overnight. The following day, I run the 230µl through a 1% gel and get a single band around 3.5 kb. I excise the band, and perform a gel purification using the Biobasic kit, add denaturant (probably guanidine hydrochloride), wait for 10 minutes for the gel to dissolve, run through column, wash 2X with wash buffer with ethanol added, elute in water.

Thanks, Max

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  • $\begingroup$ Could you share the size of your bands with us and give a bit more details about your current purification method! $\endgroup$ – Bez Dec 3 '14 at 19:42
  • $\begingroup$ The plasmid is about 3,500 kb. I incubate an overnight culture of E. coli, miniprep the following morning, elute into water. I end up with 13 ug of DNA in 200 uL of H2O. I add the Pme I (10uL) and the CutSmart buffer (23 uL) from NEB, incubate overnight. The following day, I run the 230 uL through a 1% gel and get a single band around 3,500. I excise the band, and perform a gel purification using the Biobasic kit, add denaturant (probably guanidine hydrochloride), wait for 10 minutes for the gel to dissolve, run through column, wash 2X with wash buffer with ethanol added, elute in water. $\endgroup$ – Max Dec 3 '14 at 19:53
  • $\begingroup$ 230uL of solution seems like a lot to put on a gel. Are you using multiple lanes and therefore putting a lot of gel through the gel extraction kit? I know they can only handle so much gel at once. $\endgroup$ – user137 Dec 3 '14 at 19:56
  • $\begingroup$ Could you please make this as an edit to your question! Why do a gel purification? you can just use a kit similar to QIAGEN gel extraction kit and do a non gel based plasmid purification and elution, using the buffers used as usual! PCR DNA clean up kits will do as well! $\endgroup$ – Bez Dec 3 '14 at 19:58
  • $\begingroup$ Good idea. I will attempt to purify using PCR cleanup. Thanks Bez! $\endgroup$ – Max Dec 3 '14 at 20:06
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Can't exactly say what problem you are facing but you can follow these steps for efficient restriction digestion and elution.

  • Never set up a digestion reaction with more than 1µg DNA per 20µl; if you require more digested DNA, then set up multiple 20µl reactions with maximum of 1µg DNA in each (500-700ng is ideal).
  • 10µl enzyme for a 230µl reaction: bad idea. Set up 10×20µl reactions with not more than 0.5µl enzyme per reaction tube.
  • Avoid overnight incubations; 4 hours should be sufficient (NEB PmeI is 10 units/µl and 1 unit is the amount required to cleave 1µg DNA in 1h in a 50µl reaction. You are adding 5 units in 20µl. Overnight incubation is too much)
  • Deactivate the enzyme after the reaction.
  • Just run a little bit of sample from one tube in the gel to see if digestion has happened. After confirming digestion simply use the PCR purification kit. You can pool your tubes.
  • Optional: Elute in Tris-Cl pH 7.5 instead of water or use warm (60⁰C) water.
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    $\begingroup$ Additional: Check the maximum DNA capacity of the columns used. If you overload them, you will drastically loose yield. $\endgroup$ – Chris Dec 4 '14 at 7:24
  • $\begingroup$ @Chris That is an important and nonintuitive problem, I once wasted a whole gigaprep kit because the protocol a former lab member gave me said to use twice as much DNA as the column could hold, then my advisor thought I could fix the low yield by loading even more DNA. $\endgroup$ – user137 Dec 4 '14 at 17:02
  • $\begingroup$ @user137 I have run into this problem as well, this is why I mention it. $\endgroup$ – Chris Dec 4 '14 at 17:13
  • $\begingroup$ @user137 note that a column can be reused. So you can use it for multiple rounds of elution for same plasmid. $\endgroup$ – WYSIWYG Dec 4 '14 at 17:16
  • $\begingroup$ @WYSIWYG How should I clean up the column to reuse it? Gigaprep kit are expensive and we only produce 1 plasmid on that scale so reusing a column should be ok for us. $\endgroup$ – user137 Dec 4 '14 at 17:29
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Why do a gel purification? you can just use a kit similar to QIAGEN gel extraction kit and do a non gel based plasmid purification and elution, using the buffers used as usual! PCR DNA clean up kits will do as well!

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