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Ligation Problems


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#1 jmmorales

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Posted 30 March 2010 - 12:13 PM

For the past couple months, my function in the lab I had just joined has been to insert a number of genes into Multiple Cloning Site vector for later ligation into a viral backbone. To achieve this goal, my predecessor (a post-doc who is no longer with us) and I have PCR'd the lot of the genes out of their template DNA plasmids, added unique enzyme cut sites to each of them in the process and inserted these genes as cDNA with A overhangs into T-overhang vectors so that we may be able to extract much higher yields from later steps (namely, gel extraction). The vector with a multiple cloning site and the gene were cut with NotI and PacI. Phosphatase was applied to the vector after digestion and before gel run. The Qiagen gel extraction kit was used to purify DNA from each of the digestions. The REs have no problem cutting the DNA of interest from the TOPO vector or linearizing vector DNA to yield appropriate sized fragments on a gel.

With this fragment, ligation was performed using T4 DNA Ligase. The reaction was set up with 50 ng of vector and varying ratios of insert to vector. I have used 2:1, 3:1, 5:1, 6:1, 8:1, 10:1. The formula I have been using to calculate the amount of insert to use is the following: Insert (ng)= ratio#x (length of insert/length of vector) x 50 ng. I have used controls such as linear vector with no ligase, linear vector with ligase and circular vector (uncut). Linear vector with no ligase comes up with one or two colonies max, if that. Linear vector with ligase results in the appearance of many colonies, which is similar to that observed using uncut vector. Incubation conditions for my ligation experiments have always been R.T. overnight with the exception of the last one; which I performed with a 1 hour incubation at 4 C and then transferred to R.T for an overnight ligation. The logic given behind this is that the temperature is too low for the enzyme to be fully activated and this promotes overhang-complimentary base pairing in the absence of ligation.

The re-occurring problem is that when I cut this ligated product with NotI and PacI to 'sequence' the reaction products from mini-prepped pDNA (after colony selection and expansion), I am only getting bands corresponding to the vector but not the insert. I have screened numerous colonies from as much as 20 from a single plate and every gel analysis indicates that no insert is present. When I ligate the insert (~600 bp) to itself, I do get bands at the 600 bp range and 1.2 kb range (incubation at 4C for 1 hr and then R.T. for two hours). What I take away from my results so far are that: (1) The RE enzymes work because digestion of the gene-containing TOPO vectors do yield fragments at 600 bp, and digestion of the MCS vector yields nice, clean bands at 6 kb (which is right by the way); (2) The ligase works because of the increased transformation rates (over 20X) observed from vector with no ligase to the vector with ligase rxn; (3) Vector and Insert DNA can be ligated to itself meaning that these DNA samples do have ends that can be ligated and that they aren't contaminated by the gel extraction process to the point that they cannot be ligated.

I have had the insert sequenced and everything seems correct. Being the new guy in this lab, I sure want to get this right but I am running out of new ideas. If you have any suggestions/ideas, it could really help.

#2 Michaelro

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Posted 30 March 2010 - 11:35 PM

For the past couple months, my function in the lab I had just joined has been to insert a number of genes into Multiple Cloning Site vector for later ligation into a viral backbone. To achieve this goal, my predecessor (a post-doc who is no longer with us) and I have PCR'd the lot of the genes out of their template DNA plasmids, added unique enzyme cut sites to each of them in the process and inserted these genes as cDNA with A overhangs into T-overhang vectors so that we may be able to extract much higher yields from later steps (namely, gel extraction). The vector with a multiple cloning site and the gene were cut with NotI and PacI. Phosphatase was applied to the vector after digestion and before gel run. The Qiagen gel extraction kit was used to purify DNA from each of the digestions. The REs have no problem cutting the DNA of interest from the TOPO vector or linearizing vector DNA to yield appropriate sized fragments on a gel.

With this fragment, ligation was performed using T4 DNA Ligase. The reaction was set up with 50 ng of vector and varying ratios of insert to vector. I have used 2:1, 3:1, 5:1, 6:1, 8:1, 10:1. The formula I have been using to calculate the amount of insert to use is the following: Insert (ng)= ratio#x (length of insert/length of vector) x 50 ng. I have used controls such as linear vector with no ligase, linear vector with ligase and circular vector (uncut). Linear vector with no ligase comes up with one or two colonies max, if that. Linear vector with ligase results in the appearance of many colonies, which is similar to that observed using uncut vector. Incubation conditions for my ligation experiments have always been R.T. overnight with the exception of the last one; which I performed with a 1 hour incubation at 4 C and then transferred to R.T for an overnight ligation. The logic given behind this is that the temperature is too low for the enzyme to be fully activated and this promotes overhang-complimentary base pairing in the absence of ligation.

The re-occurring problem is that when I cut this ligated product with NotI and PacI to 'sequence' the reaction products from mini-prepped pDNA (after colony selection and expansion), I am only getting bands corresponding to the vector but not the insert. I have screened numerous colonies from as much as 20 from a single plate and every gel analysis indicates that no insert is present. When I ligate the insert (~600 bp) to itself, I do get bands at the 600 bp range and 1.2 kb range (incubation at 4C for 1 hr and then R.T. for two hours). What I take away from my results so far are that: (1) The RE enzymes work because digestion of the gene-containing TOPO vectors do yield fragments at 600 bp, and digestion of the MCS vector yields nice, clean bands at 6 kb (which is right by the way); (2) The ligase works because of the increased transformation rates (over 20X) observed from vector with no ligase to the vector with ligase rxn; (3) Vector and Insert DNA can be ligated to itself meaning that these DNA samples do have ends that can be ligated and that they aren't contaminated by the gel extraction process to the point that they cannot be ligated.

I have had the insert sequenced and everything seems correct. Being the new guy in this lab, I sure want to get this right but I am running out of new ideas. If you have any suggestions/ideas, it could really help.



Hi
To my opinion, You should cut your MCS vector with each enzyme alone to make sure that the sites are intact for each enzyme.
I guess that the interval betweenrestriction sites inside the multiple cloning site is too small to be detected on double digestion.
Therefore, You cannot be sure your vector was really digested with both enzymes.
Good Luck
Michael

#3 georgiadave

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Posted 31 March 2010 - 10:28 AM

If you are cutting with both NotI and PacI then you should not get any transformants from adding ligase to the double digested vector since the two enzymes are not complementary. You are not getting complete digestion with both enzymes. For each enzyme do a digestion per your protocol, purify, ligate, transform - also transform the digested plasmid without ligase. This will tell you which enzyme isn't cutting and verify that the restriction sites are not damaged.

Also, on NEBs website (here) it states that NotI requires 5-fold more enzyme to cut supercoiled plasmids vs. linear DNA. NotI also comes as NotI-HF.

Both enzymes are heat inactivated @ 65degC for 20 min - make sure to do this, it always improves my efficiency.

Hope this helps.




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