Ligation question.

[Darwin420]

Ligation question.

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Post by Darwin420 » Sun Feb 07, 2010 1:17 am

Hey guys,

I am currently in a 3rd year molecular lab and I am conducting my first ligation next week.

I already digested my fragment of interest however I made a silly error.

For my ligation, I am also required to run a gel for the fragment of interest. I was suppose to set aside 1.5 microlitres of DNA with loading dye in a separate tube, where it will be ran on a gel. The remaining solution of DNA is the DNA I am going to ligate.

HOWEVER, I am an idiot and put the loading dye to all my DNA, even the DNA I am suppose to ligate.

Will the dye effect my ligation process?

like intuitively I would say that it wouldn't effect the ligation process... it doesn't really react with DNA, that is why it is safe to add to DNA when we run it on a gel.

However, has any one done this before? lol...

what's your insight? do you think my ligation will go fine?

[JackBean]

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Post by JackBean » Sun Feb 07, 2010 8:59 am

Hi,

you can use any of kits for DNA purification.Either some for purification after PCR or run electrophoresis with all the DNA and then cut it out and purify.
Or use just phenol:chloroform, that could work too, but I don't guarantee.

[Darwin420]

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Post by Darwin420 » Sun Feb 07, 2010 6:13 pm

Yea...but, what if I just added my solution with the dye and tried to ligate...do you think that would really mess up my ligation though?

[Darwin420]

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Post by Darwin420 » Sun Feb 07, 2010 6:21 pm

the reas why I ask this is because I don't have the lab time to to purify the DNA from the loading dye.