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4 Tips to Avoid Cloning Deletions
Here are steps you can take to increase the odds of generating correct clones.!--h2>
PCR amplicons are commonly used for cloning. Primers made from synthetic oligonucleotides often contain a small percentage of incorrect sequences which, combined with ligation and cloning errors, lead to a small percentage of clones that contain random, incorrect bases. Occasionally, researchers observe the same error repeated in many or all of their clones. This is usually a single-base deletion near the ligation site. The precise mechanism for these errors is unknown. However, in most cases, the issue appears to be unrelated to oligo synthesis based on the following evidence:
Observations from Cloning
While there are no clear rules that predict when and where such errors will occur, the deletions tend to occur near ligation sites in areas of high secondary structure, particularly at the ends of predicted hairpins that close loop structures, and at the termini of short sequences identical to those in the E. coli genome. However, these may not be the only regions that produce deletions. We have also observed that two identical plasmid vectors containing nearly identical sequences may behave differently; one may generate the required clone while the other consistently generates clones containing deletions.
Resolving Cloning Deletions
There are steps you can take to increase the odds of generating correct clones.
While these suggestions do not guarantee that cloning will be successful, they can improve the success rate and may even speed up cloning by addressing the correct source of the deletions.
Adam Clore, Ph.D. is a scientific applications specialist at Integrated DNA Technologies.
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