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Ligation


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#1 TanyaJ

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Posted 11 April 2011 - 05:42 AM

Hi guys,

I have been trying to ligate a 7.9kb insert X with 7.0kb vector Y both digested with PacI. One has Kanamycin resistance gene and other Hygromycin so I select them on Kanamycin/Hygromycin plate. However, I am not getting any colonies even though I have tried digesting the insert overnight. Once I did get a clone but it was the complete vector X (which is about 10.5kb) instead of just the 7.9kb. Since the insert and vector are of same size and the probability of self ligation is very high, I am not sure what ratio to use for ligation which will increase the canges of getting the desired clone. Please suggest.

#2 Jacques

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Posted 13 April 2011 - 06:47 AM

Hi guys,

I have been trying to ligate a 7.9kb insert X with 7.0kb vector Y both digested with PacI. One has Kanamycin resistance gene and other Hygromycin so I select them on Kanamycin/Hygromycin plate. However, I am not getting any colonies even though I have tried digesting the insert overnight. Once I did get a clone but it was the complete vector X (which is about 10.5kb) instead of just the 7.9kb. Since the insert and vector are of same size and the probability of self ligation is very high, I am not sure what ratio to use for ligation which will increase the canges of getting the desired clone. Please suggest.



Tanya,

For starters, PacI is an 8 base cutter, which with experience, does not always linearise all the DNA properly. So ensure you have nicely linearised products Don't know whether you only precipitated the digestions or used a kit to clean directly, but I'd rather excise the linear band from the agarose. Do you only have PacI to clone with?
The fact that you obtained a 10.5 kb product is worrying. Are the regions of homology between these two fragments or the the E. coli genome (the is if you are using a rec positive strain) That could be the reason for obtaining a smaller product and/or for not getting the desired recombinants. With regards to ratio. These two are virtually the same size. Although there are these nice formulas for calculating molar masses etc, eyeballing DNA on a gel works best. Run the two linear fragments alongside one another and visually determie the concentrations by estimating whether their fluoresce is the same or the one is say, half as bright. Then set up your ligation using equal amounts of each. If the are the same intensity, ligate for instance 1 ul of each. Also, use enough DNA, using to little DNA is one of the most common mistakes. If you set up a 10 ul reaction and the bands are very bright, use 1 - 1.5 ul of each. If they are faint use 4 ul of each and transform everything.

Regards

#3 mahrak

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Posted 13 April 2011 - 09:23 PM


Hi guys,

I have been trying to ligate a 7.9kb insert X with 7.0kb vector Y both digested with PacI. One has Kanamycin resistance gene and other Hygromycin so I select them on Kanamycin/Hygromycin plate. However, I am not getting any colonies even though I have tried digesting the insert overnight. Once I did get a clone but it was the complete vector X (which is about 10.5kb) instead of just the 7.9kb. Since the insert and vector are of same size and the probability of self ligation is very high, I am not sure what ratio to use for ligation which will increase the canges of getting the desired clone. Please suggest.



Tanya,

For starters, PacI is an 8 base cutter, which with experience, does not always linearise all the DNA properly. So ensure you have nicely linearised products Don't know whether you only precipitated the digestions or used a kit to clean directly, but I'd rather excise the linear band from the agarose. Do you only have PacI to clone with?
The fact that you obtained a 10.5 kb product is worrying. Are the regions of homology between these two fragments or the the E. coli genome (the is if you are using a rec positive strain) That could be the reason for obtaining a smaller product and/or for not getting the desired recombinants. With regards to ratio. These two are virtually the same size. Although there are these nice formulas for calculating molar masses etc, eyeballing DNA on a gel works best. Run the two linear fragments alongside one another and visually determie the concentrations by estimating whether their fluoresce is the same or the one is say, half as bright. Then set up your ligation using equal amounts of each. If the are the same intensity, ligate for instance 1 ul of each. Also, use enough DNA, using to little DNA is one of the most common mistakes. If you set up a 10 ul reaction and the bands are very bright, use 1 - 1.5 ul of each. If they are faint use 4 ul of each and transform everything.

Regards





hey guys
I have the same problem only my vector is pTZ57 R, which is app. 2800 bp in lenght.
I wonder if cutting the off agarose and purify this fragment would decreas the ligation efficiency.
has any one worked with long ligation kit(takara)?
tnx




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