I have made competent cell of E.coli DH5α with CaCl2 method. This is the following method:
1. Grow the cell on 50 mL Luria Bertani Broth until OD600 between 0.3-0.6
2. Aliquote the cell culture until 1.5 mL into mini centrifuge tube
3. Incubate on ice 10 minutes
4. Centrifuge the culture at 13,000 rpm 4°C 1 minute
5. Discard the supernatant and then resuspension the pellet with 0.5 mL of cold CaCl2 0,1 M
6. Centrifuge the culture again at 13,000 rpm 4°C 1 minute
7. Discard the supernatant and then resuspension the pellet again with 100 μL of cold CaCl2 0,1 M
After the competent cell is ready, I want to transformation the cell with heat shock method:
1. Add 5 μL of ligation product into 100 μL competent cell
2. Incubate on ice 30 minutes
3. Move the tube to waterbath at 42°C for 45 seconds and then move into ice bath for 2 minutes
4. Add 900 μL Luria Bertani Broth into the tube
5. Incubate at 37°C 120 rpm for 1 hour
6. Grow into Luria Bertani Agar - Ampicilin 50 mg/mL medium
After used that method the result is negative (there's no one growth). Is there any mistake of that method?
Have you got a positive control? Many commercial cells come with a pUC19 positive control (it's so common and people rarely use all of it, so ask another lab if you haven't got them). Or you can use an ampicillin resistant plasmid generated from another project.
Also, I wouldn't spin your competent cells at 13,000 rpm. You may be killing them. 3,000 rpm is enough. Please see link for more details.
http://bitesizebio.com/23336/am-i-damaging-my-e-coli-by-spinning-at-high-speeds/
Hi Johan,
The protocol seems all right. Did you check if your competent bacteria are growing without ampicillin added to your plates? It looks like your transformation did not work or another possibility, that your ligation product is not controlled by an ampicillin resistance gene, but another antibiotic?
Have you got a positive control? Many commercial cells come with a pUC19 positive control (it's so common and people rarely use all of it, so ask another lab if you haven't got them). Or you can use an ampicillin resistant plasmid generated from another project.
Also, I wouldn't spin your competent cells at 13,000 rpm. You may be killing them. 3,000 rpm is enough. Please see link for more details.
http://bitesizebio.com/23336/am-i-damaging-my-e-coli-by-spinning-at-high-speeds/
Hi, I agree with Arthur, I have different protocols for chemical or electrical competent cells and I always centrifuge at 3000 rpm for 10min at 4°C (in a protocol max centrifugation is 5000 rpm for 10min at 4°C). So make again your competent cells and pay attention that all the passages are on ice (for example when you will made cells aliquotes put, previously, the empty tubes on ice).
Hi jo, I also agree with arthur, you should have positive control.Yeah it cost more money of course. You can use another plasmid which have amp resistant gene for + control.
Make sure your ligation process is optimal. I prefer to use lower temperature in doing ligation (4 degree celcius overnight) than higher temp.
Have you check your comp cells before u use it? You can streak it on the lb agar. If it don't grow, maybe your cells are already broken. Thus you don't have a result when you use it for transformation
Hello
Its unbelievable.
I think one of your chemical compounds is corrupted. Kike ligation product.
Cheer
Hi Johan,
I agree with previous answers.
Maybe you should calculate the transformation efficiency of your competent cells
with a another vector prior transformation with your ligation product.
If you have no colonies or the efficiency is too low,
you'd probably need to make competent cells again.
Or maybe the problem are not your competent cells but your ligation reaction;
using a positive control during this step is recomendable.
Hi Johan,
I never go straight into using freshly made competent cells before doing a test transformation with a known amount of plasmid. After the transformation, I calculate the transformation efficiency (method: https://www.neb.com/faqs/1/01/01/how-should-i-calculate-the-transformation-efficiency-c3019). Only after I see that the competence of the cells is satisfactory, I will proceed with using these cells for my ligations.
Bests
And, of course, there are online calculators for everything now-days: http://www.sciencegateway.org/tools/transform.htm
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