The BP Recombination Reaction
The BP reaction is the first recombination reaction desigend to insert our gene of interest (in this case the PCR picked ras) into an Entry Clone. This Entry Clone will be used later for shuttling the gene into the Expression Clones.

Below you can see the original pDONOR201 and the resulting pDONOR201Ras that emerges following recombination. During recombination the blue fragment of pDONOR201 containing the toxic ccdB gene is replaced by the ras encoding fragment, and the L recombination sites are formed. Observe the changes in the plasmid's size following recombination (4750 bp to 2274 bp).

B1RASB1  + 
 
You have performed the BP reaction and transformed the DNA mixture to DH5a cells. In addition, you transformed other DH5a cells with  a mixture of pDONOR201 + a linearized vector containing a control gene flanked by the appropriate recombination sites. (This vector is  supplied by the manufacturer.) This system serves as a positive control.  You also transformed additional DH5a cells with pDONOR201 alone. These cells serve as a negative control.
Here are your results:
Ras Reaction
Positive Control
Negative Control
+ Kan
+ Amp

Suggest possible interpratations. (Choose between possible and wrong.)
Check ALL answers.
  1. The colonies of the ras reaction on the ampicillin plates resulted from a low efficiency BP reaction.
  2. Few colonies on Kan plates resulted from insufficient incubation time of the BP reaction.
  3. The PCR product used for the BP reaction was contaminated with low levels of the non-specific products (bands 2 and 4). 
  4. The PEG precipitation procedure didn't proceed properly, and consequently caused the formation of primer-dimers.
  5. attB-PCR primers have a mistake in the attB1 or attB2 sequences.
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