Hi, 1) T here are three reasons why the number of white clones is so much lower

Question

Hi,

1) There are three reasons why the number of white clones is so much lower than the number of blue clones (blue: 34,980 white3,785) the first reason is because the vector will religate to itself very efficiently in the absent of alkaline phosphate treatment give two reasons why white colony is a lower number? ( clue: Look at the ligation efficiencies of the vector only and vector-insert reactions. Secondly, look at the sizes of the respective plasmids (vectors Vs recombinants).

2) consider (blue colony:132 and the white colony is 0 ) in which vector is prepared for additional cloning. how would you do the experiment if you were given an insert bearing a gene that was back-to-front that is the restriction ends on the insert were in the reverse order to the vector EcoRI/BamHI vs BamHI/EcoRI?

Explanation / Answer

The reverse order of the vector will produce clones with anti sense inserts, where expression of protein will be a problem. Blunt end can be generated and then it can be cloned to provide proper orientation of inserts. The concentration of T4 ligase and DNA should be taken into care during blunt end ligation.

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