Question
Asked 24th Jun, 2013

Can someone help in regards to a problem with low efficiency of competent cell preparation?

I am using the CaCl2 method for competent cell preparation but recently I am facing low efficiency of transformation in new competent cells despite several tries. I always test them by doing transformation of approximately 1 ug of plasmid DNA that used to fill the plate but now I am only observing approximately 20 to 30 colonies each time!

Most recent answer

20th Jun, 2017
Arjun Chauhan
Dr. Yashwant Singh Parmar University of Horticulture and Forestry
Yup very true sir Michael, i got full densely covered colonies for the pure plasmid. Also, when i spread only the comp cells, it gave rise to lawn type growth.
Now, i think i gotta optimize the ligation process. 👍 Thanx for the suggestions.

Popular Answers (1)

24th Jun, 2013
Daniel M Cohen
Spark Therapeutics, Inc.
For home-made competent cells, I always use the Hanahan method. I'd strongly recommend it. Some simple factors that can strongly affect your protocol are
1) dirty glassware. Even small amounts of soap can mess up E. coli growth. Autoclave your glassware in water to clean it prior to growing your cells and
2) always use freshly streaked E. coli and never let the cells overgrow in liquid phase prior to harvesting them. It is critical to catch them in early log phase.
6 Recommendations

All Answers (18)

24th Jun, 2013
Colin Churchward
Imperial College London
CaCl transformation is always variable as you're working with a biological system. It could be any number of factors so you need to troubleshoot. Look at what you've changed recently. It could be the a new batch of media that you are using or the CaCl. Also, don't discount that it's your DNA that’s off and not your cells. If using a new plasmid prep it may have more salt in it which will affect it or too many freeze-thaw cycles. I'd start by preparing a new plasmid prep and changing your media used to grow the cells and the media used for the out-growth. Good luck!
2 Recommendations
24th Jun, 2013
Mostafa Modarresi
Change your E.coli strain!
Use fresh bacteria culture for your study
2 Recommendations
24th Jun, 2013
Tomas Chrudimsky
The Czech Academy of Sciences
how many colonies do you actually need for downstream applications? 1? 5? 20 is enough :)
from my experience-the keystone is low temperatures after CaCl2 addition (work on ice and preferably in cold room) and good starting material (commercial competent cells).
1 Recommendation
24th Jun, 2013
Daniel M Cohen
Spark Therapeutics, Inc.
For home-made competent cells, I always use the Hanahan method. I'd strongly recommend it. Some simple factors that can strongly affect your protocol are
1) dirty glassware. Even small amounts of soap can mess up E. coli growth. Autoclave your glassware in water to clean it prior to growing your cells and
2) always use freshly streaked E. coli and never let the cells overgrow in liquid phase prior to harvesting them. It is critical to catch them in early log phase.
6 Recommendations
24th Jun, 2013
Rahul Chougule
Unichem Laboratories
Use Rubidium Chloride for preparing competent cells
24th Jun, 2013
Nikos Vasilakis
University of Texas Medical Branch at Galveston
Use RbCl, complete protocol below:
Solutions:
1.5xYT
B1: 0.01M MOPS, 0.01M RbCl pH=7.0
B2: 0.1M MOPS, 0.05M CaCl2, 0.01MRbCl pH6.5
Cells:
glycerol stock of MC1061 cells
Media:
1.5xYT
In 1.0 liter
5g NaCl
6.5g Yeast extract
13g tryptone
to make agar add 4.0g bacteriological agar powder to 300 ml 1.5xYT
1. Inoculate the bacteria (1-2 colonies) into ~30-50 ml of 1.5xYT. It should grow in the shaker to the density OD600~ 0.6-0.8.
2. Transfer the flask on ice for 5 min. All the next procedures should be performed on ice.
3. Pour it to 15 ml tube. You need 3ml of culture per sample. Each 15 ml tube can be used for up to 4 samples.
4. Pellet bacteria 3000 rpm 10 min in the cold room.
5. Aspirate the media by Pasteur pipette.
6. Add to each tube ~5 ml of cold B1. Vortex tubes to suspend the cells.
7. Repeat steps 4 and 5.
8. Suspend the bacteria in ~5 ml of cold B2. Incubate 15 minutes on ice.
9. Repeat steps 4 and 5.
10. Suspend the bacteria in B2. Total volume should be 200 μl per sample X the number of samples. Do not Vortex, suspend by pipetman.
11. Aliquot the suspension to separate 15 ml tubes (200 μl/tube), add 3 μl of DMSO and plasmid sample (less than 20 μl).
12. Mix well but do not Vortex.
13. Keep on ice for 30 minutes.
14. Heat shock (45 seconds 43.5oC).
15. Keep on ice for 3-5 minutes.
16. Add 3 ml 1.5xYT to each tube. Incubate 30 minutes in the shaker at 37o.
17. Repeat steps 4 and 5.
18. Suspend the bacteria in ~50 μl of 1.5xYT by pipetting (pour few ml of 1.5xYT into separate tube and use this media for your samples).
Transfer suspension to Petri dishes with 1.5xYT agar with appropriate antibiotic. Spread it on the surface until agar will become dry. Incubate dishes at 30 oC or 37oC overnight.
1 Recommendation
24th Jun, 2013
Michael J. Benedik
Hamad bin Khalifa University
While I agree the RuCl2 method is a bit better, you should still get excellent results from CaCl2 method, we use it often in my lab. The reasons I have seen that gives low transformation are: 1) strains (some E.coli strains just don't transform well); 2) cells harvested at the wrong density. It is important not to let the cultures overgrow; 3) cells not maintained on ice after CaCl2 treatment. Throughout the preparation of competent it is critical that the cells remain chilled, this includes using a refrigerated centrifuge; 4) harsh treatment, if you vortex the CaCl2 cells you kill many of them. Resuspend and mix gently.
2 Recommendations
28th Jun, 2013
Kimjolly Lhouvum
Indian Institute of Technology Guwahati
Lithium Chloride method is the easiest, fastest and most efficient for transformation of plasmids less than 6kb. You just need 10 ml culture for 10 eppendorfs of comp cellls and requires only one centrifugation. Try out once.
24th Jul, 2013
Amir T. Marvian
Technische Universität München
Thank you all. But I think the problem originate from the plasmid either, because its length is approximately 7kb which decreases the efficiency of transformation. Do you have any other suggestion for this particular problem?
24th Jul, 2013
Michael J. Benedik
Hamad bin Khalifa University
a 7kb plasmid is not that large that it should be a problem. The efficiency will be a bit reduced but not dramatically so. Are you really using 1ug of plasmid? That is vast excess and might even be inhibitory, about 5ng is saturating in a typical transformation.
3 Recommendations
20th Jun, 2017
Arjun Chauhan
Dr. Yashwant Singh Parmar University of Horticulture and Forestry
Michael Sir, how bout the 10 kb plasmid.
20th Jun, 2017
Michael J. Benedik
Hamad bin Khalifa University
It might be somewhat lower but you should still get high efficiency. If your plasmid DNA is good and the cells are good then you should still get thousands to tens of thousands colonies if not more. What happens when you use a different plasmid as your control, that will tell you whether it is the cells or the plasmid. 
20th Jun, 2017
Arjun Chauhan
Dr. Yashwant Singh Parmar University of Horticulture and Forestry
 Sir why is so that when i am transforming with a confirmed plasmid i m getting pretty much colonies after transformation, but when i m using the ligated product i m not at all getting the colonies. Is it due to the ligation problem or what?
20th Jun, 2017
Hailiqiguli Zunuer
University of Camerino
I am having the same problem, i transformed my ligated plasmid to competent cells, but not getting my colonies at all, my competent cells are not very competitive i would like to have some idea of optimizing the transformation with lower competency cells...thanks a lot....
1 Recommendation
20th Jun, 2017
Michael J. Benedik
Hamad bin Khalifa University
The problem is that ligation is itself quite inefficient for the most part, unless you really work hard to optimize all the conditions. Frequently you might only see a few hundred colonies after transformation of ligated DNA, and that is using highly competent cells. If your cells have 10-100 fold reduced efficiency then you might see almost no colonies. A lot of people make the mistake that when they do a control transformation with pure plasmid DNA, they see a few hundred colonies on the plate and think all is fine. However with pure uncut plasmid you should see thousands or ten's of thousands of colonies, the plate should be nearly covered with colonies. 

Similar questions and discussions

Any tips and tricks to increase transformation efficiency of chemically competent cells?
Question
8 answers
  • Azreena JamahariAzreena Jamahari
Hi there, 
I have made several stocks of chemically competent DH5alpha and JM109 using calcium chloride method for cloning purposes. However, after transforming pUC19 into these cells, their transformation efficiency will never exceed 10^5 CFU/ug. Is there any tips to increase the transformation efficiency in terms or during preparation of the competent cells or transformation procedure? The method I used for making the competent cells and transformation are as follows:
1. Culture 1 colony of DH5alpha/JM109 in 5ml of LB broth for overnight at 37 degree
2. Transfer 2ml overnight LB culture and incubate at 37 degree celcius until OD605= 0.35 - 0.4 
3. Transfer 40ml of culture into ice cold sterile 50ml falcon tube. Chill the culture on ice for 5 mins 
4. Centrifuge the falcon tube at 1000xg for 5 mins at 4 degree celcius
5. Discard the supernatant and resuspend the pellet with 20ml of 75mM CaCl2 with 15% glycerol. Incubate on ice for 5 mins 
6. Repeat step 4 and 5 
7. Centrifuge the culture in 50ml falcon tube at 1000xg for 5 mins  at 4 degree celcius
8. Discard the supernatant and resuspend the pellet with 4 ml of 75mM CaCl2 with 15% glycerol.
9. Aliquot 100ul of the cells into microcentrifuge tube and snap freeze using isopropanol bath. 
Transformation: 
1, Place the competent cell on ice 
2. Add 1ul of 10ng pUC19 plasmid into the tube containing the cell. Flick the tube gently to mix the cell and DNA, Incubate on ice for 20 mins 
3. Heat shock at 42 degrees for 30 seconds 
4. Immediately incubate on ice for 2 mins 
5. Add 800ul of SOC into the tube containing the cell and incubate at 37 degree for an hour 
6. Spread the culture on LB agar containing 100ug/ml ampicillin

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