Hi all,
I performed electroporation with bacterial plasmidic extract for the first time to confirm that the plasmids carry resistance genes. Nevertheless, it didn't work. I had no colonies on the selective plates. Not even the positive control worked.
Here following I describe the protocol I followed to make electrocompetent cells and electroporation.
I made electrocompetent cells with DH5a cells as it follows:
I diluted 2 ml of overnight culture into 200 ml of 2X TY broth.
I grew them shaking at 37 C for 2 hrs.
I pelletted the cells 2 min at 4000 rpm at 4 C
I resuspended the cells in chilled sterile water, pelleted as above, repeated the wash step with water twice.
I resuspended cells in chilled 10% glycerol, centrifuged for 5 min at 4000 rpm at 4 C.
I resuspended the cells in 10 % glycerol – For 200 ml starting culture I resuspended in 2 ml of glycerol. Then I did 100 ul aliquotes.
For the electroporation:
I added 2 ul of plasmidic extract to 100 ul cells and then placed in 2 mm electroporation curvette on ice
I electroporated using 2.5kV, 25 uF, 200 ohms.I added 1 ml 2X TY media, shaked 1 h 37 C then plated on 2ug/ml meropenem, 2ug/ml meropenem and 100 ug/ml Ampicillin.
My positive control was PUC19 plasmid that confers the resistance to Ampiciln, but no colonies were present on the 100 ug/ml Ampicillin.
Can you suggest me what went gront?
Thank you very much
Hi Silvia,
Your description of the production of competent cells and the electroporation looks fine.
You could try LB plates with only Amp100 and even LB plates without any antibiotics - just to make sure that your cells are still alive after electroporation.
Have you checked the concentration and quality of your plasmid DNA? At least the positive control DNA could be checked by transformation into chemically competent cells.
Does your system displays information about the conducted electroporations e.g. duration? Maybe something went wrong at this point.
Good luck!
Hi,
I did another attempt and this time I lowered a little bit the concentration of Ampicillin from 100 to 50 ug/ml for the plate selection of my positive control (that is the plasmid PUC19 that confers the resistance to ampicillin). This time my positive control worked (or at least I think so since I have colonies on the 50 ug/ml Ampicillin plates) in spite of not having colonies for the electroporation with the Klebsiella plasmids. What could it have gone wrong this time? Could it be possible that the DH5a cells do not acquire or loose the Klebsiella plasmids just after the electroporation? Could it be due to the fact that they are two different bacteria and the host cells do not reproduce these plasmids?(anyway they are both Enterobacteriaceae)..if yes, why does this happen? Do you have other ideas and suggestions on what happens and how to overcome these issues?
Hello Silvia, in electroporation the quality of DNA is very important make sure it is purest as impure DNA or any salt may cause arcing during electroporation. Try using SOC broth with 0.4 % glucose as carbon source for the recovery post electroporation and add the SOC broth as quickly as possible, this might help you. Lastly keep everything chilled which is taken as a normal precaution during electroporation.
Good luck
Initially (in the hands of inexperienced) electroporation can be finicky. Have you tested chemically competent cells?
The efficiency of transformation may be lower using chemically competent cells when you compare the two methods (both optimized quite well) but the method is more reliable in terms of always working well.
Overall, the protocol seems ok. However, there are a couple of steps that affect the competency of the bacteria:
1. After dilution of the overnight bacteria in a pre-warmed fresh media, I usually grow the bacteria to OD 600 = 0.4 - 0.55
2. For the electroporation of the plasmid, the positive control was ~ 10-100 ng of plasmid depended on the efficiency of the bacteria
3. The the electroporation, the condition that I use 1.66 kV, 25 uF, 100 ohm, for time constants between 2 and 8 msec, in 1 mm gap curvette
Thank you very much all of you. Since my positive control is currently working anyway, but not my plasmidic DNA test (that of Klebsiella), could this due to the fact these plasmids are large and maybe they get lost by the host? Is it something that can happen?
I was also wondering (in addition t o my previous answer) if I need to treat this DNA in someway after the plasmid extraction to make it suitable for the electroporation (e.g.Do I need to linearize it?). All I am doing is to take 2 ul of plasmid DNA extracted from Klebsiella and mix it with the electrocompetent cells for the electroporation. The plasmid DNA is in water. I don't know if I am missing some step.
Hello Silvia,
DH5alpha should be fine, they have the deoR mutation that allows propagation of large plasmids due to higher expression of deoxynucleotide producing genes. What size are we talking about? 10 kb should be fine with any cloning strain. I also did up to 24 kb, it is possible.. sometimes it may help lowering the growth temperature to reduce the metabolic burden.
You don´t need to treat the DNA any further than column-purifying it and redissolve in desalted water or Tris-buffer.
Can you visualize your plasmids on an agarosegel? Don´t solely trust the Nanodrop for plasmid concentration, always check on a gel. The Nanodrop also gives you no information about the integrity, which is very important, since linearized DNA has a much much lower transformation efficiency in E. coli, since it´s very unlikely that it is repaired fast enough before the cells start dividing.
Also check the time constant in your apparatus and maybe do a test with desalted water in a cuvette to get the minimal resistance value. If your cells are washed correctly and your DNA is pure, your transformation should be in the same range (1-2 ms difference are no problem).
If you have enough material, try more plasmid DNA or purify again and elute in smaller volumes for higher concentrated. Also as suggested above try to grow the cells on plates without antibiotics.
Good luck!
Flo
Hi Florian,
thank you very much for your reply. Yes, I have a gel, I can show you. The plasmids I am trying to transform in DH5a cells are in the black circle. How can I get information about their integrity? How do I understand if the bands I am looking are the supercoiled or linear DNA? Thanks
Hi Silvia,
the small band is strange. If you have a normal plasmid prep, you sometimes get three bands (explained in the article below), but if your plasmid is huge your bands should be higher. Have you a sequence of your plasmid? If so, take a single-cutter and linearize your plasmid. Compare to your undigested plasmid, in theory in the digestion lane you only should have one band! I don´t know the size of your ladder, but it is pretty unusual for the different plasmid forms to migrate this far from each other.
If you have no sequence, just do a test restriction with a common high-frequency cutter (EcoRI, EcoRV, BamHI, HindIII etc) and sum up the separate fragment sizes. This should give you at least a hint, if you´re dealing with the right plasmid (of course you would have to know the total size for this approach).
Good luck!
http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
Thank you Florian. I don't have the sequence actually. The ladder I used is 1kb. Sorry for my questions, this is something completely new for me. I am trying to get familiar with these approaches. So, are you suggesting that the small band I see is a second plasmid?Is it possible that I see just two or one form of plasmid instead of three?
i have a question about electroporation with gene pulser xcell, i electroporate huh7 cells , with170 v,40ms 4mm cuvette, dmem (without fbs and antibiotics) as electroporation buffer, but i get very low transfection efficiency. my question is , should i use pre-warmed complete medium( dmem+ 15% fbs) without antibiotics(pen/strep1%)? does antibiotic have effect on transfection efficiency? thank you
- Badges
- Science method