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In Vitro transcription/Translation

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2 replies to this topic

#1 saad



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Posted 05 May 2009 - 09:07 PM

Hi All
I have used TNT coupled In Vitro transcription/Translation to produce fragments of protein complex to test protein interaction.
I did big mistak when I set up the reactions in put them in incubator for some reason I didn't run the incubater at 30 degree instead I left it at 20-25 degree C for 2 hours. after that I explored the problem then I changed the temp to 30 degree for just 20 mins then I run a SDS PAGE
I folow the instruction of manufacturer for everything just the temp
found more than one bands and different proteins with wrong size
any Idea appreciated.

Thank you in advance

#2 mikew



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Posted 07 May 2009 - 03:09 PM

How did you analyze your bands?
Western? S35 labeling?
This makes a huge difference.

#3 waichun



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Posted 12 May 2009 - 08:14 AM

first as mentioned above how did you detect your proteins?

lower temperature may probably slow down your reaction but not damage it. it should be alright. the best way to verify it is doing it again at 30 and you will see if anything different.
multiple bands may be due to your construct which may not be abnormal. first did you see your full-length product with correct size, probably the strongest band also?
when i translate my construct some other bands always show up which I won't see in empty vector control and negative control. it turns out they didn't affect my downstream experiment so I don't bother it anymore.

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