Lanes 2 and 3 from the BP reaction (see the right panel) resulted in the insertion of the ras gene into the Entry Clone. This vector can now be used as a shuttle vector that can transfer the ras gene into a variety of Destination Vectors that are used for expression. The various expression vectors contain different promoters for expression in different systems such as bacterial cells, insect cells and mammalian cells. They also vary in their possibilities of attaching different tags to the gene product for easy detection and purification.This is illustrated below:

You have chosen to insert your Ras gene into an expression vector suitable for expression in bacterial cells. You have chosen pDest14 that contains no additional tags and will allow the expression of your native gene. Below you can see the LR recombination reaction between pDONOR201Ras  and pDest14 and the resulting pDest14Ras.
 

Entry Vector	Destination vector	Resulting Product  (Expression Vector)
2774 bp Kan resistance	6422 bp Amp resistance	5145 bp Amp resistance

You have performed the LR reaction and transformed the DNA mixture to DH5a cells. In addition you transformed other DH5a cells with pDest14 alone. These cells serve as a negative control. Here are your results:
 

	Ras Reaction	Negative Control
+ Amp

1. The small colonies on the negative control plate result from a contamination of the original vector from which the Ras containing PCR product was obtained.
2. The small colonies on the negative control plate result from mutations in the ccdB toxic gene.
3. The ras reaction LR plates contain small colonies due to mutations in the ras gene that made it more toxic to bacterial cells.
4. The large colonies on the ras plates contain the ras gene inside the expression vector.

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