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How to clone insert with one sticky and one blunt end into vector?


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#1 Gambling Gamba

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Posted 15 August 2012 - 03:21 AM

Hi,

I do have a 1.3 kb insert digested with Bsp120L and Bgl II, the vector (7.3 kb) has been digested by Not I which is compatible with the Bsp120L restriction site. I have run all constructs on a gel, excised the linearized vector and the insert. After gel extraction (Qiagen Kit) I did phosphorylise the vector with SAP and did the first ligation step with Rapid Ligation Kit (Roche) in order to ligate the sticky ends. I set up several ligation reactions in order to have enough amount of DNA to excise from the gel. I cut out a 8.6 kb band from the gel though the amount was really low after having done the gel purification again. I filled the remaining sticky end of the vector with Klenow, stopped the reaction with EDTA and heat-inactivation and performed the second ligation. Transformation didn't show any colonies on my plates. Two questions:

1) I have transformed digested vector only as well and there are still many colonies on my plate which I can't explain. The vector has been clearly digested and I ran the DNA a long time on the gel. I never had this before.

2) Do you have a better protocol for this kind of cloning? Is it necessary to do gelpurification after the first ligation? I also could see other bands ( 4 in total) but I was thinking to try the quick & dirty way since I haven't been successful and the amount of DNA is very low. Of course I will receive several unwanted ligations but I hope that if I will screen several colonies I will be lucky with finding the wanted constructs.

Thank you!

#2 MacXP

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Posted 15 August 2012 - 07:04 AM

Hi,

Let me see if I get your problem right: you have difficulty ligating a 1.3Kb insert into a 7.3Kb vector. You still got colonies transforming the digested empty vectors.

1. The digestion was certainly not complete, even though you see a difference on the Agarose gel from the undigested control. There maybe a certain amount of vectors that hadn't been digested, which may not be visible on a gel. Still this amount is sufficient to give many colonies.

2. My suggestion is either you switch to a different vector, choose some different restriction sites, or, I'd prefer to use only one restriction site. Do a single digestion-ligation. Even it has a possibility that the insert may inversely ligated into the vector, that will only be a problem of genotyping more colonies. You still have 50% chance. Choose a good restriction site if you decided to use single digestion.

3.Is there a way that you do this cloning using gateway system? Much easier, faster and less problems. We barely do the digestion-ligation now in our lab when it comes to cloning.

Hope it helps.




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