When ordering gBlocks, one must be mindful that the desired DNA does not exceed the maximum size of 2kb. In the case where the gene of interest is close to, or longer than, 2,000 base pairs (for example, the PirB gene is >2,700 base pairs in length), it is necessary to find an appropriate cut site such that 2 (or more) gBlocks may be ordered, and subsequently ligated to reconstruct the whole gene.

The process of identifying and selecting an appropriate cut site within a gene is as follows:

  1. Find the midway mark of the gene. For PirB, this would be around base pairs 1,350 - 1,360.
  2. Identify the 4bp sequence for the Q1 cut site.
  3. Scan the middle region of the gene for this sequence (consider +/- 100bp from the midpoint). For Q1 in PirB, look for 'AGGT' between bases 1,250 to 1,460.
  4. If found, then this is an appropriate cut site: use as the ligation site when constructing pENTR (the entry plasmid).
  5. If not found, repeat the procedure for Q2, then Q3, and so on, until an appropriate cut site is found.

Once an appropriate cut site is located, split the gene into two files, and order each "half" in a separate gBlock.

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