Final answer:
You might be losing more vector than insert DNA during RE cloning due to the cloning process which includes various DNA handling steps; the inability of plasmid vectors to house larger inserts compared to phages or YACs; and inefficient amplification or purification steps.
Step-by-step explanation:
You may be losing much more vector than insert DNA during RE cloning for several reasons. One common issue is the relative size difference between vectors and inserts. Vectors such as plasmids have a limited capacity to accommodate DNA inserts, often not exceeding 1000 base pairs. When working with large genomes or long DNA sequences, other vectors like bacteriophages or Yeast Artificial Chromosomes (YACs) can incorporate larger fragments, reducing the number of clones you need to screen. This is significant when considering that a mammalian genome consists of billions of base pairs.
Another potential reason for the loss could be the multiple steps involved in cloning, such as digestion with restriction enzymes, gel purification of vectors, phosphatase treatment to prevent self-ligation, and the ligation process itself, all of which can lead to the loss of DNA. It’s important to optimize each step to minimize losses and improve the efficiency of cloning vectors.
Moreover, when using PCR amplification during cloning, vectors might undergo less significant amplification compared to the target DNA, as seen in quickchange mutagenesis, leading to a disproportionate loss of vector DNA. Additionally, inadequate removal of template DNA can also contribute to the problem, which stresses the importance of thorough DNA purification steps.