About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.
Recently, I perform a number of pflB-focA gene knock-out using lamda RED recombination in Escherichia coli strain W1485.
But I have a hard problem.
Below is my experiment:
1. PCR amplify linear fragment from both pKD3 and pKD4
Purify PCR product by gel.
2. Make target W1485strain maintaining pKD46 by growing at 30°C with 10 mM L-arabinose (E. coli was induced for just 1 h before harvesting)
3. Transform E. coli using Calcium Chlorid.
4. Plate the transformation culture on LB plates supplemented with chloramphenicol (8ug/ml) or kanamycin(40ug/ml).
Here is the results:
1. I can get several dozen colonies from plate with kanamycin, but cann't get any colony from chloramphenicol.
2. I cann't amplify anti-kanamycin gene from these anti-kanamycin strain.
Can anyone tell me what could be the problem.
During RED recombination, is electroporation necessary?
Thank you Dr. Jackbean
I have put these colonies to both kanamycin plate and kanamycine LB liquid medium.
They can growth on these medium. I purified their genome DNA, and cann't amplify anti-kanamycin gene
This confuse me.
Yes, I say this because I do a control experiment:
Same primers, same PCR conditions. The only exception is that the templete of control experiment is PKD46.
Sorry, I didn't exactly express my opinion.
I do two control experiments:
1, Amplify isocitrate lyase (using primer pairs A), to test whether the genome templete is OK.
2, Amplify anti-kanamycin gene using PKD46 as templete (using primer pairs B), to test whether the PCR system is OK.
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