A cloning experiment was being perfomred where the C. elegans unc-54 gene had un

Question

A cloning experiment was being perfomred where the C. elegans unc-54 gene had undergone PCR, than restriction enzyme digest, after which it was ligated into a plasmid vector Puc19. after ligation it was transformed into a group of E.coli where two screening techniques were undergone, ampicillin resistance and a blue white screening., aafter leaving the plates for 24 hours to cure the next day all that was seen was blue and white colonies of E. coli, A gel miniprep was undergone where 3 white colony and 1 blue colony had there DNA purified and ran on a gel electrophoresis, and that is what is seen here, im not sure about what is significant about or how to interpret this? going from left to right wells are Ladder, white colony, white colony white colony, blue colony, and Ladder THANKS ALOT

Explanation / Answer

In priniple white colonies represents the bacteria which are transfored and blue colonies the non transformed. When you amplify unc-54 gene and ligated into plasmid vector Puc19, after transformation you can also check whether the colonies contains the particular plasid y restriction enzyme digest. You have to look on the plasmid vector Puc19. map and where you have ligated the unc-54 gene. After this you can also see which restrction enzymes you have used to digest the plasmid vector after minipreparation. If the transformation is positive then after digestion you will get back the desired size bands on the gel. However, if the pasmid is not transformed or not ligated properly, the bands will be of different size than expected. Now look at the gel, the blue colonies shows a band at around 4500bp. Now, the Puc19 vctor size is around 2686bp , so together with Puc19 gene it will give aorund 4500-500 bp plasmid. Now, at the blue colonies they will have only the vector around 2686bp size( although in gel it looks around above 3000bp). so in case of blues colony the band is present below the white colonies, which indicates in blue colony you have only empty plasmid

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