1'. Wrong - the blue color of all the colonies indicates that the lacZ gene remained intact. This means that no incorporation of BHR1 gene has occurred.
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2. Possible but unlikely - usually the incorporation is not that efficient. Possibly, you forgot to add X gal and or IPTG to the plate, and so you cannot distinguish  between positive and negative results.

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3. Wrong - not adding all the antibiotics should increase the number of surviving colonies. The lack of colonies may be due to killing the cells at the time of electroporation during bacterial transformation. Alternatively, you have added too much antibiotics.

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4. Right - you should now isolate and characterize some of the white colonies, since they should contain the recombinant viral genome with the BeHappyReceptor gene ( ).

You chose six different white colonies and restreaked them in order to obtain single isolated colonies. Results are shown to the right. 
  
In order to screen for the insertion of our gene into the viral genome, you now conduct a Colony PCR assay for one colony from each streak (), using oligos derived from the LacZ gene sequences flanking the transposition sites. Results are shown to the right.
  • The expected size of the fragment containing the gene in the disrupted LacZ  is ~3.6 Kb. 
  • The expected size of the undisrupted LacZ is 300bp.
Analyze these results and determine which DNA should be used  for viral production?

A. Colony A

B. Colony F

C. Colony D.

D. Either colony A, B, C or E.

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

A. Correct, but this is not the most accurate answer,

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B. Wrong - colony F contains a hetrogenous population of both recombinant and native viral genomes.

How can  an originally white  colony produce a mixed population following restreaking?
Sometimes, the transposition occurs only during the formation of the bacterial colony on the plate and not during the previous step of post-transformation incubation. The white appearance of the original colony masks some blue bacterial cells that did not undergo transposition. Restreaking may now reveal these cells and give rise to blue and white colonies.
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C. Wrong - although the original colony appeared white, this is a homogenous population of bacterial cells that did not undergo transposition and ontains the native LacZ-undisrupted viral genome. Sometimes, when the density of the original colonies is too high, the local concentration of the Xgal substrate in the plate is depleted and is insufficient to induce the expected blue appearance of this colony.

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D. Right - each of these colonies represents a successful  event of transposition. You may safely use each of these colonies for DNA preparation to be used at the next step - viral DNA transfection into SF9 insect cells for production of Baculovirus.

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