Hi everyone?
I'm doing my thesis about Promoter regions in some jasmonate genes on cassava (M. esculenta). I have any questions about the DNA cloninig...
Is possible lose some pair bases (bp) of the promoter's sequence?. I mean, If I have my amplified product (promoter) of 1000 pb by PCR (normal), will I get the same sequence of 1000 pb (promoter) on the DNA cloning?
Under what circumstances get lose pair bases of a sequence?
Does get lose some DNA pb being inserted on a plasmid?
On the other hand, occurs the same in the DNA sequencing?
Excuse my english (is my first post) :)
If you design your PCR and cloning experiments well then you don't expect to lose bp of the promoter sequence you want to clone. You will only lose a few bp on each side of your PCR product after restriction Enzyme digestion (these are restriction sites added to the 5' of forward and reverse primer). This small change in the size of your PCR product will not be detectable on an agarose gel.
Here is a link about designing PCR primers for cloning
https://www.neb.com/protocols/0001/01/01/primer-design-e6901
Unintentional deletions, insertions and mutations can always happen during cloning, but you should observe such events only rarely. If there are many clones containing the deletion you speak about, then it is very likely that there is something wrong with the experimental design (wrong primer design, introduction of an additional restriction site, etc.). On the other hand, if the cloning results with only a few viable clones, and all of them have some deletion or a mutation, it is likely that your insert is for whatever reason toxic and the clones are now selected in an unwanted manner (those that have the mutated insert survive).
Good luck!
I agree with the previous answers.
Also considering the primers were designed correctly and you used suitable PCR conditions for amplification, then the amplification efficiency depends on the polymerase used to amplify your product. For long targets that contain GC- or AT-rich sequences, it is sometimes difficult to amplify by general DNA polymerases.
First, I recommend checking your target gene in the cloned plasmid by reading the DNA sequence. If the deletion is found in all the checked colonies then try to use different primers or use more efficient polymerase such as Tks Gflex DNA Polymerase.
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