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Help with ligation

ligation transformation

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2 replies to this topic

#1 fmoafi



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Posted 18 January 2012 - 02:55 AM

Hi everybody,
I have double digested my pSUPER vector with bglII and Xho I, I run on the gel and the digestion appeared to be working well. but when I do ligation followed by transformation, I do not get any growth. I am sure nothing is wrong with ligation procedure because I did the transformation with an old digested vector and it did work there and I got colonies. It appears that something is wrong with the vector I made, it just doesnt work, I do not know if it is due to damage by UV while I was extracting the vector from gel. By the way I used the restriction enzymes from two different companies can any one help?

#2 joy123



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Posted 28 February 2012 - 02:23 PM

I formerly experienced failure with ligation, and it was probably because the insert was too long. I suggest you confirm your ligation step first before transformation. You may do it by cutting with enzyme and check the band size.

#3 Makosad



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Posted 01 March 2012 - 01:27 AM

try to heat the vector and insert separately at 50 degrees for a couple of minutes, that helps sometimes to arms of vectors free and accessible for efficient ligation.

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