Hi, I want to insert 130 bp DNA into 3000 bp vector, however I could not obtain colonies. I used 50 ng insert DNA and also tried to increase insert amount but still I could not observe any colonies. Is there anyone who tried to insert small DNA (nearly 100 bp) and experienced this problem? If so, can you explain your solution?
Small fragment like 100 bps insertion should be easier in general. People normally insert small fragment by primers PCR mutagenesis method. I assume you get your 100 bp fragment by synthesis two oligoes instead of a PCR product. To assemble a DNA fragment you need to mix equal amount of complementary oligoes in a tube, heat it at 95 degree for 5 minutes, then incubate at 70 for 10 minutes to allow them renature into a proper DNA fragment before you use. Infusion reaction itself also often contacts problems which you may need to take account.
I extended oligos by PCR then tried to insert PCR product. PCR mutagenesis method may not be useful for me because I need to remove certain sequences in the vector with digestion before the infusion. Thanks for the advises.
Hi, I work with fragments of 200bp and apparently the low efficiency of recombinant colonies is normal. I recommend you:
take extra precautions when handling your vector as temperature changes can break it down, you probably need to change it for a new tube.
Check the order in which you make your ligation mix.
Check the concentration of antibiotic you use and whether the cells are still viable.
I hope it helps, greetings!
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