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pvdQ Protein Expression

 

To express the pvdQ protein, it was cloned into the pET21a (+) expression vector. The Pet21a (+) was digested with NotI to yield a linearized 5443bp fragment. Dephosphorylation of the vector fragment using calf intestinal alkaline phosphatase was carried out to prevent relegation. Then, the pvdQ-B0015 gene in the pSB1AK3 vector was also digested using NotI, yielding the desired fragment size of 2,400 bp.

 

 

The two digested fragments were then ligated. Since both fragments yielded NotI sticky ends, performing restriction digestion to check the orientation is essential. The ligated product of correct orientation when digested with ScaI and DraIII would yield fragment sizes of 660, 2014, and 5237 bp. When in correct orientation, digestion with SalI would yield bands that are 952 and 6959 bp. The fragments ligated in an incorrect orientation would yield fragments of 660, 1277, and 5974 bp when digested with ScaI and DraIII. When digested with SalI, it would yield fragments of 1542 and 6369 bp.
 

 

From the gel photo, it can be deduced that Sample 3 yields the desired band sizes, and therefore produces the correct ligation product.