Gel Electrophoresis of Ttr (PCR) within the pCR2.1 cloning vector and the digest
ID: 214053 • Letter: G
Question
Gel Electrophoresis of Ttr (PCR) within the pCR2.1 cloning vector and the digest product of the plasmids.Lane 1, 100 bp Ladder, Lane 2, EcoRI control, Lane 3, EcoRI 1, Lane 4, EcoRI 2, Lane 5, EcoRI 3, Lane 6, EcoRI 4, Lane 7, Drall II control, Lane 8, Drall II 1, Lane 9, Drall II 2, Lane 10, Drall II 3, Lane 11, Drall II 4, Lane 12, 1 Kb Ladder.
Can you explain what is happening in the gel electrophoresis in terms of Ttr gene inserted in antisense and sense orientation.
It appears that the restriction digest did not work at evey site due to short incubation time. Circular DNA could have been uncut so the linear DNA became more compact and supercoiled DNA run farther down the gel. Or if looks smeary then plasmids were not completely clean. Can you indicate what is happening for each band. I am confused.
No inser in the vector plasmid: EcoRI 284 and 300 bp and Dra II 351 bp
EcoRi had insert cut in 2 places to have 450bp and 650 bp for the antisense direction
Dra ll had insert cut in 780 bp/1100 bp. At 350 bp cut + 50 bp end of insert for Dra ll . Then the insert cut is 370 bp for the antisense oreintation
The insert will make the full lenght of the plamid 5000 bp -370 bp=4630 bp
So Dra ll has 370 bp and 4630 bp in the antisense oreintation.
Drall has 4170 and 830 bp for the sense orientation
Please explain in detail to understand such as explaining what is happening and which band give an antisense and sense orientation based on banding size. Please show work to understand the gel
Explanation / Answer
1. In cell electrophoresis, the Trt genes get inserted randomly. The sense orientation is more thicker like a band and the antisense spread in an irregular manner. The place where the DNA becomes more thickly sedimented is because of the flow of photons due to the process of electrophoresis. The place where the photons are concentrated in the sense plasmids and the place where the plasmid is spead is irregularly is the disease.
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