What next in the antitoxin knockout endeavour?

OK, time to end my two three? four? weeks of sulking because my experiments won't work...

Where was I?  (... must consult my notebook)

I was trying to resolve two issues.  The first was why ligation of my phosphorylated PCR fragments did not produce a plasmid that could transform E. coli to AmpR SpcR.  The second was why my plasmid preps were not producing any plasmid, from cells that I was quite sure contained a high-copy-number plasmid.

This second issue was compromising my ability to investigate the first issue, so let's deal with it first.

I was doing my plasmid preps using Econo-spin spin columns (from Epoch Life Sciences) and column reagents we had made up ourselves.  This is much cheaper than using spin-column kits from Qiagen or Sigma.  But the columns and reagents were quite old, and I was not even sure that I was using the right volumes of the reagents.

Here's what I was doing:

The basic procedure is to start with a version of the standard alkaline-lysis procedure:  

1. Pellet cells and resuspend in a neutral buffer containing EDTA
2. Add 0.2M NaOH +1% SDS.  The SDS will lyse the cells and the high pH will cause the base-paired DNA strands to separate, 'denaturing' both chromosomal DNA and the plasmids.  Each plasmid's two DNA strands will be looped together because they are interlocked circular molecules.
3. Neutralize the NaOH and make the SDS insoluble by adding a low pH potassium acetate solution.  The two plasmid strands will regain their base pairing returning the plasmid to its normal configuration.
4. Centrifuge the tube to pellet the cell debris, including the chromosomal DNA and most of the SDS.  The plasmid DNA and various soluble components remain in the supernatant

In the old-fashioned procedure the supernatant is extracted with phenol and then chloroform, the DNA (and RNA) is precipitated with ethanol, and the pellet is resuspended in TE buffer (often with RNase A added to degrade all the RNA).

With a spin column the supernatant is instead placed in the top part of the column (see figure above, from Perkin-Elmer), and spun through it.  The DNA sticks to the filter membrane in the base of the column, and all the unwanted soluble material washes through and is discarded. The DNA stuck on the membrane is further cleaned with one or two washes of a special solution containing ethanol, and then the DNA is eluted from the membrane into a clean tube using a small volume of TE buffer or water (usually 50 µl).

The kits typically are expensive (Qiagen: $1.35 per column). They include all the reagents, but the reagent recipes are kept secret.  The same columns work for a number of different DNA-purification procedures, but you need to buy a different kit for each to get the specific reagents used in each procedure.

Epoch and other budget suppliers will sell you just the columns (Epoch, about $0.40 each) and provide recipes so you can make your own solutions for all the procedures.  But as I said, our Epoch columns were old (several years?) and I wasn't confident that my hand-written notes specified the correct volumes of reagents to use.  We had the sheet with the reagent recipes but I couldn't find one with the protocols.

Anyway, I did a test.  With replicate samples I used our homemade reagents to do the alkaline lysis steps 1-4 above, and then finished some sample with a spin-column cleanup and some with phenol extraction and ethanol precipitation.  The column samples had some chromosomal DNA contamination but no plasmid and no RNA.  The phenol-etc samples had no chromosomal DNA and no plasmid but a lot of RNA.  Bummer.

So I did another test, this time adding to the two treatments above samples where I did the alkaline lysis steps using reagents specified by an old non-column protocol, followed by the old-fashioned phenol extraction and ethanol precipitation steps.  This time I did get some plasmid from the old-style prep, but again none from either prep using the column reagents.

So this suggests that the column reagents or volumes are at fault.  I checked online, but found a slightly different set of protocols, with some reagent names we didn't have the recipes for.  I contacted Epoch and they kindly sent me a new recipe sheet.

Epoch also provided this advice about column storage and regeneration:

If you have access to cold room you can store the column in a cold room. Or if you still have room in your refrigerator which does not go through defrost automatically you can also store your column there. We can also ship you some "moisture pack", most conveniently with your next order, which can be sealed in the plastic bag. Finally you can apply 20 ul of 0.2N NaOH to the old column 30 minutes before you do miniprep. This will regenerate the membrane to the maximum bidding capacity.  

So...  I suspect my preps didn't give plasmid because I was using the wrong volumes of the column alkaline-lysis reagents volumes, not because the cells didn't contain plasmids.

How does this affect my thinking about the rest of the work?  Might some of the colonies I discarded have contained a desired plasmid?  I'll put that in the next post.