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ironman's Content

There have been 7 items by ironman (Search limited from 24-September 19)


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#24942 PCR problems on a ligation product

Posted by ironman on 21 May 2009 - 06:50 PM in PCR, RT-PCR and Real-Time PCR

What length of overhang do you have between each of the three pieces before you try to ligate them to each other??
How long, what temp do you use of your ligase reaction?
How do you prepare the fragments for the ligation, after PCR amplification?
Do you know if your ligase is working? Test it on some DNA ladder; if you don't get the ladder shifting up with a 20 minute, RT reaction, the ligation step is failing.

I have about 5bp overhang for the three pieces.
Usually I allow the ligation reaction to run overnight at 4 degC. If I run it for 2 hours, the reaction is at 20-25 degC.
After PCR amp, I run the mixture in agarose gel electrohoresis. I use Qiagen Gel Extraction kit to extract DNA from the gel band with the expected size.
I believe the ligase and buffer are working because we have used it in other reactions with no problem at all. And a band corresponding to the right size of the three pieces ligated together is showing up. I know this is not conclusive, that's why I tried to amplify each of the piece using the ligated product as the template and I got all the three pieces amplified.

Thanks.



#24648 PCR problems on a ligation product

Posted by ironman on 18 May 2009 - 06:42 PM in PCR, RT-PCR and Real-Time PCR

Hi! I have a ligation product composed of three pieces and which is about 4kb in size. I only get a small amount of this ligated product and so the next approach is to amplify the ligated product using PCR. The problem is, I can't seem to amplify the linear DNA fragment using the outside flanking primers. I have tried a nested PCR, in which I tried to amplify each of the piece and I got PCR products, of about the right size for each of my pieces. What seems to be the problem here? Thank you very much for the help.

We need your reaction conditions before we can help...


Thanks. The PCR reaction consists of:

5 microL buffer
4 microL dNTPs
1 microL primer1
1 microL primer 2
37.75 microL H20
0.25 microL ExTaq
1 microL template (my ligation product)

The PCR running conditions are:

94 degC- 2mins
94 degC- 30 secs
50 degC- 45 secs (annealing temp and time- i have rechecked this)
72 degC- 4 mins (extension time; polymerase activity- 1 kb/min)
4 degC

Ran for 30 cycles...

Thank you.


I'd just like to add that I have also tried clone the ligated fragments into a vector, but I was not successful. The thing is, when I try to amplify each piece in the ligated product (just to check their presence), I am getting PCR products, a possible indication that they are present in that DNA fragment. Is it possible that they have not really ligated. The ligation reaction was gel-purified. I extracted the band which corresponded to the approximate size of my expected ligated product.



#24645 PCR problems on a ligation product

Posted by ironman on 18 May 2009 - 06:18 PM in PCR, RT-PCR and Real-Time PCR

Hi! I have a ligation product composed of three pieces and which is about 4kb in size. I only get a small amount of this ligated product and so the next approach is to amplify the ligated product using PCR. The problem is, I can't seem to amplify the linear DNA fragment using the outside flanking primers. I have tried a nested PCR, in which I tried to amplify each of the piece and I got PCR products, of about the right size for each of my pieces. What seems to be the problem here? Thank you very much for the help.

We need your reaction conditions before we can help...


Thanks. The PCR reaction consists of:

5 microL buffer
4 microL dNTPs
1 microL primer1
1 microL primer 2
37.75 microL H20
0.25 microL ExTaq
1 microL template (my ligation product)

The PCR running conditions are:

94 degC- 2mins
94 degC- 30 secs
50 degC- 45 secs (annealing temp and time- i have rechecked this)
72 degC- 4 mins (extension time; polymerase activity- 1 kb/min)
4 degC

Ran for 30 cycles...

Thank you.



#24632 PCR problems on a ligation product

Posted by ironman on 18 May 2009 - 04:53 PM in PCR, RT-PCR and Real-Time PCR

Hi! I have a ligation product composed of three pieces and which is about 4kb in size. I only get a small amount of this ligated product and so the next approach is to amplify the ligated product using PCR. The problem is, I can't seem to amplify the linear DNA fragment using the outside flanking primers. I have tried a nested PCR, in which I tried to amplify each of the piece and I got PCR products, of about the right size for each of my pieces. What seems to be the problem here? Thank you very much for the help.



#19517 Help in Liagtion Procedure

Posted by ironman on 20 March 2009 - 03:00 PM in Molecular Biology

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!



#19409 Help in Liagtion Procedure

Posted by ironman on 19 March 2009 - 04:39 PM in Molecular Biology

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?



#19367 Help in Liagtion Procedure

Posted by ironman on 19 March 2009 - 12:27 PM in Molecular Biology

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!




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