What you are getting are probably false positives. Do the following:

1-When preparing electrocompetent cells for this purpose you have to concentrate them well at least 300 fold concentration therefore start with a large volume of the culture for the cells at around 200ml.

2-In some cases increased amount of DNA seems to help therefore add the maximum possible amount you can without exceeding 10ul if you are using 100ul of cells for electroporation (it is argued that 10ng should saturate the electroporation, however, I usually got better results when use 1-1.5ug of linear DNA).

3.After electroporation quickly add SOC to your cells and allow them to recover overnight without shaking at room temperature. You can then plate on LB kanamycin the next day (I recommend you use lennox LB or luria LB since the standard high salt miller LB will decrease viability after electroporation). With certain antibiotic resistances you will get poor or no colonies if you recover for 1 hour at 37C therefore overnight recovery will help.

Just an additional note unless you know that knocking out the gene will cause your cells to be sick or you are using a strain that is slow growing already, a lack of growth after 1day of plating after overnight recovery most likely indicates that the electroporation have not worked and any colonies showing up after that are most likely false positives.

Hanna Alalam , can you please share what antibiotics need a longer recovery time? And thanks for the insightful answer above!

Hello Alejandro Martin I only tested a limited subset but the results as follows:

Kanamycin and chloramphenicol are a hit and miss so sometimes with the 1 hour recovery with SOC at 37C you will not get any colonies, therefore, overnight recovery at room temp without shaking is recommended (if you look at the original paper they stated that they have proceeded this way for colonies that failed to grow when recovered only for one hour).

I also tested erthromycin and in this case I only got colonies from overnight recovery (doubling the recovery time to 2 hours did not help).

I hope you find this helpful.

Best,

Hanna

Hello Manuella Silverio, we have been using the Datsenko system for several years. We have had problems especially with new isolations (low efficiency of obtaining mutants). Now we are using CRISPR-Cas9 with much better results. If you have interest we can do some work in collaboration.

Regards

Mariano

Hanna Alalam , Thanks for such helpful suggestions!

1. After overnight recovery at room temperature without shaking, how much volume do you plate? Do you pellet down entire volume and plate everything?

2. Also, sometimes people suggest to use a lower concentration of antibiotic in this plate for faster growth and later when colonies appear transferring it to higher or actual antibiotic concentration. Does it help?

Hello Tias Saha , I usually use the entire volume as you have said. As for the 2nd part the lowered antibiotic concentration might help (some people believe that since you integrate a single copy a lowered antibiotic concentration might help especially if you are not using overnight recovery since in normal transformations plasmids are multicopy therefore are able to produce a higher amount of the resistance in shorter timer), however, the overnight recovery is usually sufficient to plate directly on the normal selective concentration. In some particular cases I tend to use half the selection concentration (this is still well above the MIC) especially for chloramphenicol, these cases are:

1-You expect a sick strain that is slow growing.

2-a strain that has another resistance already integrated in the chromosome (since double selection usually reduced the growth)

Hope this helps!

Hi again!

Thanks Hanna Alalam for the answer.

I have another doubt,

1. After electroporation and recovery ( bei it, 24 hours or 4 hours) there are difference in sizes of colonies. We got colonies within 12 hours but some were too small (they didn't grow even after 24 hours) and others were normal sized which grew bigger with increasing time. Why would there be two distinct sized colonies with difference in growth rate?

2. What are the possibilities which can give rise to false positive colonies? Even if the amplicon was DpnI digested and gel purified, can there still be false positives? If so, how?

Thanks everyone in advance!

Hello Tias Saha ,

It is simply that the small ones are false positives than can be due to multiple reasons:

1-If you are selecting on reduced concentration of your antibiotic at the beginning you might get a low level resistance that is enough to grow small colonies on the reduced antibiotic concentration and then when you transfer to the high concentration they will die.

2-The antibiotic in the plate is degrading hence there is a lower actual concentration than you think there is.

3- (This one is a bit a stretch but I guess it could still happen)The recBCD system is inhibited with the expression of RED proteins therefore linear DNA should be stabilized, you might get some expression of your resistance marker without actually integration and if your are unlucky enough you might be able to see pinpoint colonies.

4- Finally DpnI digestion in not enough to prevent positives due to plasmid transformation (that was what they observed in the original paper that why they used pir dependent plasmids as templates). If your source of template is not a pkd plasmid or other pir dependent plasmid I would suggest you switch to those. The reason why dpnI+gel purification might not be enough is because the digestion will not be complete and the template plasmids are usually small therefore the supercoiled form will co-migrate with the amplified sequence and would be impossible to distinguish.

Best of Luck!

I also think you are getting false positives. The most important step in this method is making sure your PCR products are of extremely high concentration. In my opinion, that is a very critical step. When you have your PCR product concentrated, run 1ul of it on an agarose gel and you should see a huge think band. That is when you know you are ready to transform your PCR product for recombination. I think chloramphenicol is a better option if possible as it has a lower background. Also use fresh plates with antibiotics to rule out any background.

Hello Manuella,

I am facing a very similar problem right now. Eventhough I have much more clones on my transformation plates than the negative controls (no DNA added). But still every clone I screened is negative. I suggest that this is due plasmid contamination or stability of the kanamycin cassette. Eventhough I purified my PCR product via gel electrophoresis, I guess (as suggested by Hanna Alalam) it would be better to use pir plasmids.

Did any of these recommendations help you? If yes, please feel free to tell me :).

In any case I will try again with the pir plasmid, a higher concentration of linear DNA and recovery over night. So far I used ca. 0.3 - 0.4 µg template, maybe it's not enough.

Warm regards

Roman

@Braira Waheed could it be possible that you are getting a PCR product with your primers because they are binding nonspecifically to some other region on your genome? Maybe you have knocked out your gene but the PCR is non-specific? Maybe you can sequence your results to find out

@Braira Wahid, maybe you could also use some other PCR primer set. For example if your target gene is 1kb, use a primer set that will PCR a region outside of the 1kb gene and then sequence that PCR product too. This way you can compare and see!

@Braira Wahid, it could happen that the antibiotic resistance cassette by which you are trying to replace the gene of interest has approximately same size as your gene, so you will not by able to resolve the difference between a mutant and the wild-type strains by gel. You can: 1) either use your current Fw and Rv primers to amplify the region of the mutant DNA and send for sequencing 2) or you can design one more primer which binds somewhere within antibiotic cassette and use this primer in pair with your Fw or Rv (in this case for wild-type there will be no band amplified and for the mutant you will get a fragment)