Hi! I worked with this plasmids, and with this strain...And i didnt have any problem to have them both in the same bacteria... Have you evaluate the eficiency of your competents? are you doing them with the corresponding antibiotic?

I electropored the pCas first at 2,5 kV in a 0,1 cm gap cuvette first, and then, after making a clon competent, the pTargetF with the same conditions.

I don`t know why are you having problems.. an you are transforming a lot (a lot!) of circular plasmid... even 1 ng should be more than enough, and you shouldnt be able to have discrete colonies after plating..

tell me more about what you are doing :)

Hi Victoria! Im glad you answered me! I really don't know why it doesn't work, today I did the same again with different voltages, 2,5 kV, 2,2 kV, 1,8 kV and 1,4 kV. Tomorrow I will see the results! I'm working with 1 mm cuvettes, in one paper they used 2 mm cuvettes with only 50 µl of cells..

so you first transformed pCas too and then pTargetF? Wasn't there any problem to make the "same" cells competent again? Did you relax them after electroporation in SOC medium too? I use Kanamycin and Spectinomycin as recommended..

I'm really excited about the results tomorrow! According to your answer it should have worked with 2,5 kV!

so my thesis is about "knock-out of the lacz gene in E coli MG1655 through CRISPR Cas 9 technlogy" and im the "first one" using the technology in our lab :) the group before struggled with the transformation of pCas, pTargetF worked very well. the eficiency of pTargetF is high like 10^3

pCas has a temperature sensitive origin and must be grown at 30 degrees C. According to AddGene it is RepA101ts. The ori in pTargetF is pBR322 (according to the map for the parent plasmid pTrc99A). So no plasmid incompatibility issues here. I've never done CRISPR! Sounds Cool!

@Victoria: it did not work! What were you grow conditions of the MG1655 with pCas? Until which OD you let them grow and how did you get them competent? What was your antibiotic concentration? Which Medium did you use and what kind of plates? What was your goal/project at the end?

@Terry: Thank you so much for helping me! CRISPR is really cool, especially when it's working! :D

Hi again!! As Terri said, in orden to make the MG16655-pCas competents I grown them at 30ºC and 200 rpm to DO 0,5. After transformation I relaxed them with LB medium -wich was the one i have, i mean, for no especial reason- and I used the antibiotic concentrarion recomended..

The goal of my project is to edit a virus genome, wich i try -unfortunately without success- to do inside Ecoli with these plasmids.These days I am changing my approach and leaving behing the two plasmid sistem (I am too the first one using the technology in my lab and it is being tricky for me too! ).

Nevertheless I belive it is a great way to edit E coli genome. You ll get there! I dont know what is gooing on with your MG16655-pCas competents, do you have experience doing competents cells? Maybe it has someting to do with that...It doesnt seems to be anything wrong with what you are doing...

Once I contact the autors of the paper with some questions...they were really nice, may be they could help yopu more...I am sorry I might not been of help. I am here anyway if you have more questions, or a fellow CRISPRCas worker :)

Good luck!! I am rooting for you!!

Hi,

I don't know if it helps, but our lab uses a BioRad Gene Pulser with a capacitance extender. We use an electroporation cuvette with a 1 mm gap.Try these settings: 1.8 kV, 200 Ohms, 25 uF. After the pulse check the Time Constant. It should be around 4-5 msec. If the pores are open too long (>5) the cell viability decreases. If the pores are no open long enough (

Hi!

@Victoria, but after growing them you centrifuged them and washed them with sterile water 3-4 times and put dem into glycerin to conserve them? Or how did you make them competent?

Did you also use the paper of Jiang about the "multigene editing.."?

Well thank you so much, I'm growing my bacteria now to death to see, if the exponential phase is really from 0,4 to 0,7.. I will check also if they are really competent with another plasmid instead of pTargetF.

I wish you a lot of luck for your project! Seems like you're standing still like me :(

@Terry: our lab has the electroporator from Eppendorf and its only possible to change the voltage, idk why.. But you're right, if they are not open long enough it's hard to get the plasmid inside. I already checked the transformation of pTargetF in my normal MG1655 with different voltages, it transformed really well from 1,4 kV up to 2,5 kV so I think that should not be a problem.. I really want to know why its not working with pTargetF into MG1655 pcas. Maybe they are really not competent ?

Hi again!!

i make my competents with a lot of ice (a lot!). A soon as the get to de DO y put them in a water-ice bath for 30 minutes, and stir them every 5 or 10 minutes. I do al the washes after centrifugation with Glicerol 10%( made with double-distilled in water, like the LB or the medium you are growing your cells). And the Glicerol 10 % should be also cold! i put the bottle in a telgopor box - i don´t know how to call it- with a lot of ice.

the eficiency of the competents really increase with this modification.

you can check this paper, it is about another strain, buy it may help..

Article Enhancing DNA electrotransformation efficiency in Escherichi...

Hello girls! I was looking for some solution and I ran into your conversation. I am having the same problem. I'm trying to use the two plasmids system and I already inserted the N20 sequence in pTarget without any problem but when it comes the first electroporation of pCas in MG1655 I get clones also in the control (no plasmid) and none of the clones in the transform ants are correct. I used the same values Victoria Nugnes mentioned, 2.5 kV in a 0,1cm cuvette and I always get 5.40 -5.60 as a result of the electroporation which should be fine. I grow them in 30°C adding also Kan and Arab, but nothing.

So I'm wondering what could be wrong in the procedure. The author gave me the plasmids and the MG1655 strain. I checked the sequence of each plasmid after extraction ad they are ok, I always use no more than 100ng for the electroporation.

Plus I designed a pair of primers with a fragment of Cas sequence around 300 bp and when I do the PCR to identify the clones, all the bands are around that size, even the control which actually don't have plasmid.

And when i grew MG1655 the first time to make them competent I grew them in 37°C with kan and I didn't get any growth.

Any other suggestions?

Hi! Giorgia Croppi what do you mean with that you already inserted the N20 sequence in pTarget ? with PCR? did you circularized it? I don`t understand because to do so you have to transform the MG1655 pcas with the linear product of the pcr..

it is really weird that you are not having any colonies in the transformant bacterias, and you are having in the control....

did you checked you competetns cels? maybe they are contaminated... For what you are telling me it looks that way.. if you spread an MG1655 competent aliquot in LB with Kanamicyn, and results in colonies you had some contamination in the competent preparation. Maybe the primers you designed are hibridating with the contamination's Dna, ( I assume you checked that they dont prime with e coli mg1655 genome...)

i would transform the plasmid they give you and start over with a new culture.. always with Kan and at 30ºC

Good luck!!