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help with ligation/transformation


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#1 whatfangz

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Posted 30 March 2009 - 11:19 AM

I originally posted this in the 'Molecular Biology' section, not having seen this section, which is more appropriate. So I'm posting again here.

I have had trouble with this transformation for over a month now. I hope someone can help me! Thanks.

I have a 1.9kb insert, double-digested with XbaI and XhoI, that I want to transform into pFastBac vector (4.8kb), also double-digested with the same enzymes. I gel extract the double digest reactions with Qiagen kit, and have a concentration of about 15-20 ng/ul for the insert and somewhere 35-60 ng/ul for the vector. I'm not sure if these concentrations are important, but I do find that these concentrations work best for generating transformants. I've also had very low concentrations before, like 5 ng/ul for the insert, that do not work as well (not sure why, since the final volume is always 20 ul, so the final concentration of DNA should be the same...).

I use the NEB Quick Ligation kit, which is a 5min RT ligation. Then typical transformation protocol is used. The heat shock is usually 45 sec at 42o. Although I have also tried the 1 min at 37o. I usually do 1:1 and 1:3 vector:insert molar ratios, and also plate ligations for vector with no insert (negative control). Yesterday's plates had nothing on the negative control or the 1:3, and about 10 colonies on the 1:1. I screened a few of them, but they are all false positives. This has happened to me before with a different insert, where there was nothing on the negative control, but quite a few maybe 10-20 on the 1:1, and nothing on the 1:3. The 1:1 colonies were all false positives, so this trend is not a good sign.

I also just ran some of my old ligations on a gel, and saw only bands that were near the top of the gel. I read about this, and it seems these may be concatemers? I guess my insert ligating to itself over and over? I'm not sure exactly how that works. Anyway, any help is appreciated.

There is a PstI site between the XbaI and XhoI sites in my vector, but my insert also has that, so I cannot digest vector without insert, without digesting everything. My colleague has suggested phosphatase, but I am not sure how this procedure works. Can anyone help??? Thanks!

#2 Dr Teeth

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Posted 31 March 2009 - 04:40 AM

I originally posted this in the 'Molecular Biology' section, not having seen this section, which is more appropriate. So I'm posting again here.

I have had trouble with this transformation for over a month now. I hope someone can help me! Thanks.

I have a 1.9kb insert, double-digested with XbaI and XhoI, that I want to transform into pFastBac vector (4.8kb), also double-digested with the same enzymes. I gel extract the double digest reactions with Qiagen kit, and have a concentration of about 15-20 ng/ul for the insert and somewhere 35-60 ng/ul for the vector. I'm not sure if these concentrations are important, but I do find that these concentrations work best for generating transformants. I've also had very low concentrations before, like 5 ng/ul for the insert, that do not work as well (not sure why, since the final volume is always 20 ul, so the final concentration of DNA should be the same...).

I use the NEB Quick Ligation kit, which is a 5min RT ligation. Then typical transformation protocol is used. The heat shock is usually 45 sec at 42o. Although I have also tried the 1 min at 37o. I usually do 1:1 and 1:3 vector:insert molar ratios, and also plate ligations for vector with no insert (negative control). Yesterday's plates had nothing on the negative control or the 1:3, and about 10 colonies on the 1:1. I screened a few of them, but they are all false positives. This has happened to me before with a different insert, where there was nothing on the negative control, but quite a few maybe 10-20 on the 1:1, and nothing on the 1:3. The 1:1 colonies were all false positives, so this trend is not a good sign.

I also just ran some of my old ligations on a gel, and saw only bands that were near the top of the gel. I read about this, and it seems these may be concatemers? I guess my insert ligating to itself over and over? I'm not sure exactly how that works. Anyway, any help is appreciated.

There is a PstI site between the XbaI and XhoI sites in my vector, but my insert also has that, so I cannot digest vector without insert, without digesting everything. My colleague has suggested phosphatase, but I am not sure how this procedure works. Can anyone help??? Thanks!


Concatamers occur when molecules of digested insert or plasmid ligate with other molecules of digested insert or plasmid. Remember that an insert with XbaI and XhoI sticky ends can ligate to another insert with XbaI or XhoI sticky ends in an opposite orientation as can digested plasmids since the ends are also compatible with each other not just for plamid/insert. If you are getting concatamers for every clone, this suggests to me that you have too many molecules of insert or plasmid or there is a problem with the ligation. It seems as if you are doing everything correctly and it sounds as if your procedures usually work for you, so I would assume that your calculations for 3 M ratios, etc. are correct also. In that case, have you tried "normal" ligations (ie. not using the quick kit)? Perhaps a 4-5 hr RT ligation or overnight at 16C. I doubt that phosphatase will help since you are not getting religation of your base vector.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley




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