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Ligation - (Jun/23/2007 )

Hi everydy blush.gif
Im trying to ligate 500bp DNA fragment into 2.7kb vector. After doing double digestion, I purified both of them.
My vector band is stronger than my insert( 2-3 times) . I dont have any idea to measure my DNA or vecor to be able to guess what ratio is approprate for my ligation.
1-What you guys suppose to do???

2- is orientation of the lacZ important? because the ECORI is one side and HIND III the other side in my vestor ( I did double digestion with those 2 enzymes) . I put EcoRI in the begining of my forward primer and Hind III in reverse primer.
I know that both enzymes produce 5-sticky ends. I did right or not???

-PUC18-

QUOTE (PUC18 @ Jun 23 2007, 10:16 AM)
Hi everydy blush.gif
Im trying to ligate 500bp DNA fragment into 2.7kb vector. After doing double digestion, I purified both of them.
My vector band is stronger than my insert( 2-3 times) . I dont have any idea to measure my DNA or vecor to be able to guess what ratio is approprate for my ligation.
1-What you guys suppose to do???

2- is orientation of the lacZ important? because the ECORI is one side and HIND III the other side in my vestor ( I did double digestion with those 2 enzymes) . I put EcoRI in the begining of my forward primer and Hind III in reverse primer.
I know that both enzymes produce 5-sticky ends. I did right or not???


Hi, a 1:3 vector to insert ratio generally provides good results in ligation. To calcualte the relative amount of vector and insert DNA that gives this 1:3 ratio, you can use the equation provided in the Promega T4 DNA ligase product insert, se url below
http://www.promega.com/tbs/9pim180/9pim180.pdf. If you know the amount of DNA in the bands of your ladder, you can determine the concentration of DNA in your samples semi quantitatively by comparing the intensity of a chosen volume of your DNA to the intensity of the band of similar size in the ladder, and obtain an estimate of the concentration oof DNA in your sample.

Your second question regarding the orientation of lacZ is not clear. Fact is if you r constructing a recombinant plasmid merely for cloning then orientation doesnt matter. However if the recombinant plasmid was constructed so as to express a particular gene, then of course the orientation of insert in vector will matter. Directional cloning, which you have done, will result in the insert being in only one orientaion in the vector and that orientation is that which was dictated by the researcher (you) based on which end of the insert the two RES are and also the location of these two RES on the vector. Of course EcoRI overhang on your insert will ligate to the EcoRI overhang of your vector and the same for the HIND III sticky ends. From the information you presented, I cannot deduce wether or not the orientation of the insert would be correct for expression.

-aneisha-

Thanks Aneisha for your tips biggrin.gif
Acyually,Im gonna do site-directed mutagenesis in the puc18 after putting that 500bp in it. Im gonna change 2 aminoacids in the insert after cloning into the puc18.
I think in this case, orientation in puc 18 should be important.So I upload the direction of the EcoRI and HindIII in the puc18 to take a look at it. As I said my forward primer (contains HindIII and my reverse primer contains EcoRI in my insert. so Im wondering if I have done the right thing or not??


In addition I had purified puc18 and my insert. Today I did double digestion with EcoRI and HindIII. I run the gel to see how stong are my bands( I didnt run those after doing double digestion) Unfortunately I found out that I lost my Insert( 500bp).!!! I dont know whu and how!!! I add EcoRI first for 3 hours and then I added HindIII for 3 hours. I dont know the reason!

-PUC18-

well 2 things could have happened.

1- the DNA was indeed lost. In which case, the probably place was when the DNA was percipitated and supernatant removed. The pellet can detach from the walls if one is not careful. So spin down the DNA after every step, including after the 70% EtOH wash. THis prevents the DNA from falling off.

2- there wasn't very much DNA to begin. THus the DNA is not lost merely hard to see. Holding the tube to a strong light, and carefully removing supernatant, should allow you to see small bumps or a light smear on the tube wall.

EcoRI and HindIII can be digested together in NEB buffer2. There is no need to do separate digest.

Does your department have a nanodrop?
There is quite a trick to accurately tell how much DNA is present in a sample, using DNA ladder of know quantity.

-perneseblue-

Thnaks for your tip
Unfortunately we dont have a nanodrop
My incubation time( incubation for the first enzyme was 3.5 hours andfor the second one was 3 hours) is ok? maybe thats the reason I lost my DNA fragment?

-PUC18-

If the DNA is free from nucleases (which it should), digesting your DNA for 6hrs will not cause a problem. If the DNA was being digested for 2 days, then maybe but certainly not 6hrs.

It probably when down the wash, or it is still in the tube.

-perneseblue-