Hello
I have tried introduce pCL55-itet which is subcloned into B. subtilis ISW1214. pCL55-itet has ampicillin resistance marker So, I incubate B. subtilis ISW1214 on TSA + 100 mg/L ampicilin medium after tranformation. After 24 hours, I have found colony on TSA + AMP 100 mg/L and I proceeded colony PCR using pCL55-itet amplication primer. Howver I couldn't get any band on colony and I got band at positive contol. Why I couldn't find any band using colony even if it is grown on TSA harboring ampicillin??
Hello Lee, it would be great to mention how your transformation was done and if it a cloning reaction. If this was a cloning experiment, it is ok to have negative colonies on the plate and a screening of many colonies is usually required to get positive ones. In the case of just one negative colony, i would suggest you check on your cloning again, do an electroporation if you initially transformed by heat shock and lastly, you should first re-suspend the transformed bacteria in antibiotic-free medium for 30 mins - one hour to allow the expression of the antibiotic resistance gene before transferring to a new plate with antibiotics.
Hope this helps. All the best!
Hello Lee Byunghoon , in addition to the answer by Ferdinand Ngale Njume you may also try to do restriction digest of purified plasmid from few colonies to make sure that your insert is actually present.
Courage!!!
Hello
My friend has done colony PCR several time, but it did not work. I mean we could see any band or could see smear (maybe the colony which was added to the PCR reaction was too much). Then I extracted the plasmid and did the PCR on DNA. So, the problem solved and I could see specific band like my positive control. Be sure that your bacteria is transformed, because has grew on selective media. Only if you add antibiotic to hot medium, the antibiotic will be inactive and the non transgenic bacteria will grow in it.
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