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Double digestion and ligation problem


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20 replies to this topic

#16 Julio-Claudian

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Posted 26 March 2010 - 09:53 PM

If you intend to clone a PCR fragment you MUST purify before cutting the DNA. Otherwise, the (still active) PCR enzymes and dNTPs will extend the 3' end of the cut DNA and destroy the cohesive site, preventing ligation.


Speaking of purification, can anyone confirm for sure that doing gel extraction (for the desired product) after digestion prevents ligation (of the digested PCR product) to the vector? What really happened? Did the whole process of extraction remove the sticky ends?

Would it be recommended to deactivate the RE followed by ligation?

#17 phage434

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Posted 27 March 2010 - 07:56 AM

If you do not inactivate the restriction enzymes somehow before ligation, the ligation will not work when recreating the restriction site. This can be useful if you are relying on ligation of compatible ends which do not re-form the recognition sequence (usually from ligation of cut fragments from different enzymes).

Heat inactivation or gel purification can be used. I try to avoid gels, primarily because they are not robot-friendly, but others have good luck with them.

#18 William Parkar

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Posted 21 April 2010 - 10:14 PM

1. an complete (never cut) vector: three bands: 4k(main), 7k, 9k.
2. all cut vector(by only one RE or two): one band at 6.1k

both above results are ok

3. re-ligase vector:



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#19 Biocat

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Posted 01 June 2010 - 02:07 AM

If you do not inactivate the restriction enzymes somehow before ligation, the ligation will not work when recreating the restriction site. This can be useful if you are relying on ligation of compatible ends which do not re-form the recognition sequence (usually from ligation of cut fragments from different enzymes).

Heat inactivation or gel purification can be used. I try to avoid gels, primarily because they are not robot-friendly, but others have good luck with them.



But if the vector (2984) was cut from another expression plasmid (5070), is it possible for ligation without gel extraction in such case?

#20 nazmunpstu@yahoo.com

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Posted 28 April 2012 - 09:17 PM

Dear Sir

I have made double restriction enzyme digestion of a Cucumber gene qith T-vector and Overexpression vector (PCAMBIA1305.1 Vecto)r with restriction enzyme NcoI-HF and BstEII (NEB) at 37 and 60 degree C temperature respectively for a total 4 (2+2) hours. After running the gel I got good band in T-vector and my gene. But the band PCAMBIA1305.1 was not good and the cut portion about 2000 bp was not clear. After extraction of my gene and overexpression vector, I made T4 DNA ligation and following everything means PCR, again double restriction enzyme digestion, which were OK. Then, I sent to company for sequencing and they told me it is multiclone. I am very much trouble with this. Please help me.

#21 phage434

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Posted 29 April 2012 - 05:08 AM

There might be three possible reasons for this:
(1) your construct has multiple copies of the binding site for the sequencing primer. You could try a different primer or examine the sequence carefully with bioinformatics.
(2) you could have a mixed population of cells. The easiest way to fix this is to dilute your plasmid DNA, re-transform, and pick single colonies showing the correct bands.
(3) very rarely, your plasmid construct could have multiple copies of the plasmid origin (usually this requires one of them to be inactive). In this case, cutting your plasmid DNA with a single cutter, re-ligating, transforming, and isolating single colonies can fix this problem. This really is a sub-case of situation (1) but is caused by there being two copies of the plasmid origin in a single plasmid construct.




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