Recombination Products When Cloning Repeat Elements

February 01, 2019

Albert Einstein College of Medicine
Genetics Department
Ph.D. Student

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Company:

New England Biolabs

Product Name:

NEB® Stable Competent E. coli (High Efficiency)

Catalog Number:

C3040I

I am trying to make DNA constructs that contain multiple repetitions of a target sequence of interest. I previously used another E. coli strain (DH5-apha) but I could not obtain the desired number of repetitions due to recombination. I purchased the NEB Stable bacteria because it is meant to be recombinase negative. I followed the protocol's instructions, growing the bacteria at 30°C for 24 hr (instead of 37°C, 16 hr) and I still got a lot of recombination in my samples. I have managed to obtain some of the plasmids I needed, but it is not as efficient as they describe on their website.

Experimental Design and Results Summary

Application

Cloning of multiple repeat sequences

Starting Material

NEB Stable cells and ligation product of plasmid + sequences

Protocol Overview

Cells were transformed using the protocol provided by NEB: Pipette 50 µL of competent cells in Eppendorff tube. Add 2 µL of ligation product. Incubate 30 min on ice. Heat shock at 42°C for 30 sec. Place on ice 5 min. Add 950 µL of NEB 10-beta/Stable Outgrowth Medium. Grow at 30°C in a shaker (250 rpm) for 1 hr. Spread 100 µL on a selection plate and grow at 30°C for 24 hr.

Tips

The only solution I found was to do many colony PCRs for each construct (transformation efficiency was good, so I have a lot of colonies).

Results Summary

I still got a lot of colonies that had multiple bands after PCR, which indicates the plasmid has recombined and the bacteria have a mix of plasmids with a different number of repetitions (see in the picture).

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

It is slightly better than DH5a or XL-1, since I did get some plasmids without recombination.

The Bad

Repeats are not as stable as they claim on the website, at least not with my sequences.

The Bottom Line

The percentage of recombination was high with my sequences, but at times I did get a construct that had the correct number of repeats and hadn't been recombined.

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