To me it seems impossible to proceed for pENTR D-TOPO cloning without the insert having CACC over hangs, I would like to know what others think about this.
You can get inserts, although with very low efficiency (an internal CACC is sometimes used). However, most people use this vector to express a eukaryotic gene. The CACC also acts as a ribosome assembly site (Kozak sequence); the most important base is the -3 position relative to the AUG initiating methionine - it should to be a purine for efficient translation to occur. The flanking Cs (and G at +4) further improve efficiency.
If you have or can get pCR™8/GW/TOPO®TA Cloning vector from nearby labs then certainly with the available primers you generate an ENTRY clone. The ORF frame with respect to "attL" sites will remain the same as in pENTR/D-TOPO. However, you need to verify the direction of the cloned inserts among few clones to get the desired forward direction clones by colony PCR or digestion.
you can add it by PCR by using the new designed primer (the same your primer but you must add CACC at the end of it ).
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