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PCR and restriction enzyme digestion


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#1 ahmadfathi

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Posted 16 February 2013 - 03:45 AM

hello, i done transformation on dh5-alpha culture and the culture is grown, when i screen the culture by performing PCR, it give positive result (band shown at expected size). however, when i retest with double digestion, there no band even for the positive control. i used 200 ng and 10U (0.5 ul) at 37oC for an hour then inactivate immediately at 65oC for 20 minute? is it my transformation is really worked? any recommendation?

#2 bob1

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Posted 16 February 2013 - 11:47 PM

If your + control didn't work - then you can't say anything about the experiment... you either need to troubleshoot the experiment until your + control works, or find a different way to get the confirmation you need.

#3 ahmadfathi

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Posted 17 February 2013 - 12:25 AM

do you means i still need redo the transformation,or just optimize the extraction for restriction enzymes digestion?some of my colleague said these happen because of insufficient concentration of DNA that need for the digestion..

#4 bob1

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Posted 17 February 2013 - 11:30 AM

I would start with getting the digestion working - that's your test. You have some evidence that your ligation is working from the PCR.

Keep this in mind - a small fragment of DNA cut out of a plasmid may not show up on a gel if there isn't enough DNA to bind the EtBr and give a visible band. So, 200 ng of plasmid may not be enough to see a cut out band, though you should be able to see the initial plasmid band.




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