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.

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!

Change your E.coli strain!

Use fresh bacteria culture for your study

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).

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.

Use Rubidium Chloride for preparing competent cells

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.

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.

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.

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?

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.

Michael Sir, how bout the 10 kb plasmid.

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. 

 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?

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....

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. 

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.