"In plate" / solid medium transformation of Acinetobacter baylyi ADP1

The following protocol is for single colony "in situ" (in plate) transformation with minimal use (1000x less) of transforming DNA.

Background

Generating transforming DNA at sufficient amounts for transformation reactions can be difficult, specially if it requires gel extraction given the low yields of this purification method. A method that requires less use of transforming DNA can be a time saver and thus serve to simplify the process of genetic manipulations.

This is a quick and simple "in situ" colony transformation method useful for transforming naturally competent ADP1.

Method:
1. Plate 100uL of a 10^-6 dilution of cells onto LB and incubate overnight - colonies should be nicely spread out.
2. Dilute transforming DNA to .2ng/uL and directly pipette (.25-.50uL) carefully up and down over a single colony. Keep plate leveled so that the "on plate" rxn doesn't run over to nearby colonies. (note: pipeting up and down is not absolutely necessary but is likely to increase transformation efficiency).
3. Incubate for another 24 hrs.
4. Pick up (with a sterile spatula or any other tool) all of the enlarged colony treated with transforming DNA and dilute on 100uL saline.
5. Plate all on selective media - optional, do negative control with untreated colony from the same plate. Also, transformation frequencies may be computed by diluting in a larger volume at step#4 and using some of the diluted culture to do a cell count plate on LB.

Advantages:
As opposed to using the standard 100ng per transformation reaction, this method allows equivalent successful transformations with 1000x times less transforming DNA. Furthermore, do to the size of the in situ (plate solid media) transformation rxn and the minimal DNA necessary, this method easily allows to saturate cells with transforming DNA, which may aid to increase transformation frequencies of troublesome transformations. Lastly, several single colony transformation rxns can be performed on a single plate without cross contamination, therefore this approach is useful as a high-throughput multiple transformation method.

"Puddle transformation"

Brian Renda routinely performed these. "Puddle transformations" are another very convenient way to transform ADP1 in solid medium. In principle, you just use 50ÁL of overnight culture and mix in the transforming DNA (this step can be done separately or in the plate) spotting the cell-DNA mixture in a non-selective LB plate to allow the transformants to grow and then scrapping off the cells to plate them in selective media. Optionally, for easier and near complete removal of the transformed bacteria, you can use a nitrocellulose membrane to grow the ADP1 spots on, then use the resuspended transformants to prepare both count and selection plates to determine transformation frequencies.

Similar to transforming in solid media as described previously, in a puddle transformation the relative concentration of transforming DNA increases, thus it can result in very high transformation frequencies (E-1 or 1-10% transformation). Since this method of transformation requires very low amounts of DNA, it is particularly useful for use with plasmids.

Procedure: Grow ADP1 in LB overnight, put 50 Ál of cells in a puddle on an LB plate (spotting it over a nitrocellulose membrane; note these are not usually sterile), add 1-25 Ál of DNA (from a prepared dilution at ~1ng/ÁL), incubate 18 hours, scrape (or just carefully lift off the membrane) and dilute (drop membrane with cells in tube with 1 mL sterile saline and vortex) and plate 100ÁL or the same amount from a 10-fold dilution. Generally, the number of cells in the 1mL saline can be expected to have the same cell concentration as those grown overnight in LB. See also notes section under Acinetobacter Transformation.

Brian Renda even suggested once: "I bet we could scale up the procedure to use an entire plate. This would work by purposely growing the culture and DNA together in a lawn, adding media and plating beads, shaking to resuspend the culture, and then bringing that forward into whatever selection step is next."

-- Main.GabrielSuarez - 10 Oct 2016


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Topic revision: r3 - 27 Sep 2017 - 21:14:11 - Main.GabrielSuarez