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Help in Liagtion Procedure


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#1 ironman

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Posted 19 March 2009 - 12:27 PM

Hi! I've been trying to make a 3-pc ligation of DNA (DNA1, DNA2, DNA3). The three pieces have approximately the same MWs. I have three separate pieces, all cut by the same restriction enzyme. I did several ligation combinations (DNA1 + DNA2, DNA2 + DNA3 and DNA1 + DNA2 + DNA3) and upon gel electrophoresis, I got the same resulting bands for the three different combinations, plus some bands from the unligated pieces. How was this possible? Of course, I expect the third combination to have a higher MW compared to the first two combinations.

Is it possible that I got DNA1+DNA2+DNA1 and DNA3+DNA2+DNA3, for the first and third combinations respectively because they had similar restriction sites.

Thanks!

#2 swanny

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Posted 19 March 2009 - 03:04 PM

Hi! I've been trying to make a 3-pc ligation of DNA (DNA1, DNA2, DNA3). The three pieces have approximately the same MWs. I have three separate pieces, all cut by the same restriction enzyme. I did several ligation combinations (DNA1 + DNA2, DNA2 + DNA3 and DNA1 + DNA2 + DNA3) and upon gel electrophoresis, I got the same resulting bands for the three different combinations, plus some bands from the unligated pieces. How was this possible? Of course, I expect the third combination to have a higher MW compared to the first two combinations.

Is it possible that I got DNA1+DNA2+DNA1 and DNA3+DNA2+DNA3, for the first and third combinations respectively because they had similar restriction sites.

Thanks!

What are the sizes of the individual fragments?
What are the sizes of the ligation products?
What were your ligation reaction parameters?
Can you show us a gel image?
Why did you use the same enzyme?
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#3 ironman

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Posted 19 March 2009 - 04:39 PM

Hi! I've been trying to make a 3-pc ligation of DNA (DNA1, DNA2, DNA3). The three pieces have approximately the same MWs. I have three separate pieces, all cut by the same restriction enzyme. I did several ligation combinations (DNA1 + DNA2, DNA2 + DNA3 and DNA1 + DNA2 + DNA3) and upon gel electrophoresis, I got the same resulting bands for the three different combinations, plus some bands from the unligated pieces. How was this possible? Of course, I expect the third combination to have a higher MW compared to the first two combinations.

Is it possible that I got DNA1+DNA2+DNA1 and DNA3+DNA2+DNA3, for the first and third combinations respectively because they had similar restriction sites.

Thanks!

What are the sizes of the individual fragments?
What are the sizes of the ligation products?
What were your ligation reaction parameters?
Can you show us a gel image?
Why did you use the same enzyme?


DNA1- 1.23 kb; DNA2- 1.3 kb; DNA3- 1.25 kb. The size of the ligation products is about 2.5kb for all the combinations. Ligation reaction was done according to the manufacturer's instructions. The same enzyme was used because of the restriction sites avaliable and to prevent enzyme incompatibilities. Is that a no-no?

#4 perneseblue

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Posted 20 March 2009 - 04:12 AM

DNA1- 1.23 kb; DNA2- 1.3 kb; DNA3- 1.25 kb. The size of the ligation products is about 2.5kb for all the combinations. Ligation reaction was done according to the manufacturer's instructions. The same enzyme was used because of the restriction sites avaliable and to prevent enzyme incompatibilities. Is that a no-no?


Since all three DNA fragment have been cut with the same restriction enzyme, there is no directionality in the ligation reaction. Any and every reaction that can occur will occur, not only the your desired reaction.

As all three fragments have been cut by the same enzyme, there is no reason why the DNA1+DNA2+DNA3 mixture should produce higher MW bands than the DNA1+DNA2 mixture or even a DNA1 (alone) ligation mix. The fragments all have the same ends and are thus equivalent.

What you want is something like this.
EcoRI-DNA1-BamHI
BamHI-DNA2-NotI
NotI-DNA3-EcoRI

Only when the end of DNA fragments are incompatible will the 3 way ligation work as the number of routes that lead to a circular molecule is sharply decreased. You won't see a difference on a gel though.
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#5 ironman

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Posted 20 March 2009 - 03:00 PM

DNA1- 1.23 kb; DNA2- 1.3 kb; DNA3- 1.25 kb. The size of the ligation products is about 2.5kb for all the combinations. Ligation reaction was done according to the manufacturer's instructions. The same enzyme was used because of the restriction sites avaliable and to prevent enzyme incompatibilities. Is that a no-no?


Since all three DNA fragment have been cut with the same restriction enzyme, there is no directionality in the ligation reaction. Any and every reaction that can occur will occur, not only the your desired reaction.

As all three fragments have been cut by the same enzyme, there is no reason why the DNA1+DNA2+DNA3 mixture should produce higher MW bands than the DNA1+DNA2 mixture or even a DNA1 (alone) ligation mix. The fragments all have the same ends and are thus equivalent.

What you want is something like this.
EcoRI-DNA1-BamHI
BamHI-DNA2-NotI
NotI-DNA3-EcoRI

Only when the end of DNA fragments are incompatible will the 3 way ligation work as the number of routes that lead to a circular molecule is sharply decreased. You won't see a difference on a gel though.


Thank you very much for the suggestion and the clarification!




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