When I was doing construction, I hope to insert a 10kb fragment into a 10kb plasmid.
The plasmid was digested by an enzyme, and the fragment has homologous arms which are the same as the digested site.
After I transformed the ligated 20kb plasmid into E.coli and purify it to do sequencing, the sequencing result showed that about 20bp of my fragment inserts into the plasmid. This 20bp is the same sequence of the primer used to colon the targeted fragment. The others are missing
There may be lots of problems for sub-cloning a 10 KB fragment into a 10 KB plasmid after restriction enzyme digestion. Did you use one or two different enzymes to cut out the insert fragment? If you used single enzyme, there will be self ligation of your plasmid that will exclude any insert, unless you performed dephosphorylation. Also, the amount of insert required is at least five times more than the insert. Finally, depending on sticky or blunt end of the restriction site, ligation conditions should be optimized. So, failure of not obtaining inserts could be due to a number of technical hurdles.
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