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Basic electrophoresis digest banding pattern analysis....

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Basic electrophoresis digest banding pattern analysis....

Postby scholesmu » Thu Apr 28, 2011 12:44 am

I want to know more about banding pattern analysis......

lane1: standard makers (HindIII digest, sizes are 23.1,9.4,6.6,2.3,0.6,0.1kb)
lane2: undigested plasmid X
lane3: plasmid X with restriction enzyme SciI by student A
lane4: plasmid X with restriction enzyme SciI by student B
lane5: plasmid X with restriction enzyme SciI and ArtI by student C


What can i conclude on plasmid X?


what reason by student A and B can make such difference?
lane 3(student A) don't digest......can it....due to "not adequate enzyme added....?
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Postby JackBean » Thu Apr 28, 2011 6:35 am

it must be cut, since it's different from line 2 ;)

ather, student B added not enough plasmid, since there are bands respective to both lines 2 and 3 ;)

And BTW you have more bands in line 1 than you should have
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Basic electrophoresis digest banding pattern analysis....

Postby whizliz » Wed May 04, 2011 10:03 am

I'm no mol bio person but why is the uncut control (2nd lane) running as two bands so far apart? Isn't plasmid supposed to run in three bands pretty close to each other?
Student A (lane 3) looks as if he has
(i) Forgot to add enzyme/added too less.
(ii) Added way too much plasmid.
The later seems more likely as the band that you have drawn is very thick compared to others. Enzyme activity is measure in ug of DNA that it can cleave; if concentration of your template is beyond the capacity of the enzyme that you have added then you will not get an expected fragmentation pattern.
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Postby JackBean » Wed May 04, 2011 10:45 am

whizliz: I have always seen only 2 bands in line with uncut plasmid.
Why do you think, that Student A did have too much plasmid in comparison with RE? (actually that's the case of student B)
http://www.biolib.cz/en/main/

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Postby crazydavid1628 » Fri May 27, 2011 6:42 am

I don't understand why the undigested plasmid has two bands.
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Postby JackBean » Fri May 27, 2011 10:51 am

because it's supercoiled. Look on Wikipedia for plasmid conformations ;)
http://www.biolib.cz/en/main/

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