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ligation problem with the insert size bigger than 4kb


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5 replies to this topic

#1 henanli

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Posted 19 July 2002 - 09:44 AM

Hi :
I met a problem with the ligation. iinsert  size is 4.7kb (digest with not-1, sal-1) vector is NCOR 7.5kb(digest with not-1, sal-1). I tried insert:vector 1:1 3:1 and 10:1 and got nothing.
the vector amount is 100ng.
anybody has any suggestion or experience?

#2 rhodadg

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Posted 29 July 2002 - 10:28 AM

Hi,

You should be able to ligate your fragment to your vector.  Are you treating the vector with shrimp alkaline phosphate?  Are both insert and vector clean? How did your ligation controls turn out? How are you transforming your bacterial cells?


#3 henanli

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Posted 11 August 2002 - 09:49 AM

my vector is CFP-C1 and digest with sal1 and not-1 then run gel to purify. Idid n't do CIP or any]thing to  it.
I transformation to DH5a.

#4 henanli

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Posted 11 August 2002 - 09:52 AM

my vector is CFP-C1 and digest with sal1 and not-1 then run gel to purify. Idid n't do CIP or anything to  it.
I transformation to DH5a.
I'm not so sure about the ratio of insert: vector.

thank you for reply me.


#5 rhodadg

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Posted 12 August 2002 - 05:11 PM

I think you may be using too much vector.  I never use more than 50ng per 10ul ligation reaction. I'm not too sure how you set up your ligations but here's how I do it.

1ul of vector (vector concentration 50ng/ul or less)
1ul 10X ligation buffer (Roche)
1ul T4 ligase (Roche)
7ul insert DNA (at least 50x's more concentrated than my vector)**
ligate for at least 16hrs at 15 degrees celcius

**I usually have my insert DNA resuspended in water so that I'll reduce arching during my transformations but I have also found that if the insert DNA is TE buffer diluting your ligation reaction 1:10 reduces arching.

Hope this helps


#6 Wotan

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Posted 13 August 2002 - 07:58 AM

From my experiences it is very important to expose the DNA very little or better NOT to UV light to get good yields of ligation-product.
For preparative purification of DNA fragments by gel-electrophoresis I use two lanes for each fragment:
one small, which is exposed to UV-light for diagnosis, and another big, which is not exposed  to UV but cutted at the position as the interesting band is located at in the small lane.
The eluted DNA then never was exposed to UV which increases the number of colonies found after transformation
ten fold.




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