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same vector but diffirent clones produce bands of different size. - (Oct/17/2006 )

i don;t know how to explain this. i have made an empty vector from the one i received from my freind. i simply cut out her insert (which i don;t want) and purify it by gel etc....i blunt end, and religate the vector, electroporate and get a lot of colonies. i miniprep some clones (colonies) are of the right size but some seem bigger even up to by 1Kb. blink.gif . i can;t get it. they are all derived from the same vector.... unsure.gif unsure.gif


Did you blunt the ends yourself by filling in or digesting away overhangs, or by using a restriction enzyme that produces blunt ends?

Weird thngs happen when cloning; I frequently see stuff that's too big or too small and ignore them in favor of the right-sized clone. Confirm by sequencing over the junction...



Could probably be vector ligating to one another?

-I love MSGs!-

what was the size of insert? if it is around 1kb, i can say the uncut vector might be dragged with the cut vector. this can happen in case your DNA is concentrated or wells of your gel is quite narrow. and in case u don't give DNA enough time to seperate during electrophoresis. so u might have transformed vectors with or without the insert. u may check this by restriction analysis using an enzyme that cuts the insert only.


thanx for the tips! i used Klenow to blunt end and ligation mix kit to ligate. i dont think vector could ligate 2 of them together that would give me much bigger product. the insert is 38bps.... laugh.gif noway to dinstinguish here is guess. and sequencing can't be done in this lab since sequencer is just sitting and not being used by anyone... huh.gif

Homebrew, i chose the one that is closest to the right one, but still i think it is little bit smaller. please tell me gel markers are not so accurate anyway. smile.gif