Questions tagged [ligation]
Is the process of covalently link two DNA ends. The process is usually done by the enzyme class of ligases.
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I accidentally plated DH5a E. coli and left them in the 4℃ for 12 hours, if I put them into the 37℃ incubator will they start growing?
I started ligation of my constructs late last night and was super tired, since I used all of my ligation mixture I would rather not have to re-ligate.
I did my transformation procedure as follows:
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modify PCR steps to include a ligation
Imagine a multiplex PCR in which after the extension step, the dsDNA is nicked and requires a ligation reaction to repair the nick before the next denaturation step. So the steps would be:
Denature
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What is the status of the cfDNA ends?
I am working on a protocol to ligate cfDNA, extracted from blood, into some vector. I have looked into the literature but I cannot find any detail about the status of the ends of the cfDNA. Are they ...
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Separate DNA fragments with very different size (oligo and lambda-DNA)
I am running a ligation reaction to one of the sticky ends of lambda-DNA.
The oligo for the ligation is 25 bp (10x excess), whereas lambda-DNA is 48 kbp. I would like to recover the lambda-DNA and ...
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Ligation without purifying insert
I am planning to insert a 45 bp sequence in a vector. After restricting my insert to create compatible sticky ends, I am finding no way to clean it up. Is there any way for cleaning this fragment ...
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Is blunt end repair and A-tailing necessary when adaptors can be custom made?
I'm planning on creating a RADseq library, which naturally involves digesting genomic DNA following P1 adaptor ligation. I've read a few protocols that require any over-hangs resulting from ...
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Can Bacteria Repair Dephosphorylated Plasmids?
I have been trying to insert a short sequence of DNA into a plasmid. I wanted to make two different versions, with the insert at different locations. I chose HinDIII and SalI sites approximately ...
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Freezing after restriction digest
Brief question:
I have performed a double digest with heat inactivated restriction enzymes with no star activity. I usually purify the DNA (PCR inserts and linearised vector) using a kit or gel ...
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What happens after the purification step in Hi-C sequencing?
I am a statistician reading an article on Hi-C, and I am trying to better understand one of the steps in the DNA isolation and sequencing process.
Since I'm a statistician, please try to avoid too ...
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How are split inteins joined?
Do they form a peptide bond? Or is it just some affinity/van der Waals interaction?
Is the mechanism enzyme catalized?
Are the residues/moieties at the join site known? Could they be introduced to ...
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Closing a Plasmid by Ligation
I have been having trouble with what should be a fairly simple ligation. I had a plasmid that I needed to cut part out of, so I designed a couple PCR primers to do that. The plasmid already had a ...
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Can someone link me to resources on the efficiency of sticky end ligation?
I really would like to know if sticky end ligation could potentially be performed with very high efficiency, and which factors influence that. However, I can't find any papers on the subject, even ...
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circularizing DNA molecules?
I have been reading about next-generation sequencing technologies that can sequence long reads. Even though the origin of my question is sequencing technologies, the question I am asking is about the ...
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What is the minimum length required for a circular DNA ligation?
What is the minimum length of a DNA molecule for the ends to come in close enough contact that they can ligate. Assume there are free divalent cations in solution. Does anyone have any idea?
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What does 5' and 3' mean in DNA and RNA strands?
What are 5' and 3' in DNA and RNA strands? Please clarify with some images and please use simple English.
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3' or 5' DNA capping protein
Does anyone know of a protein/molecule that can be attached to one end of the DNAs temporarily ? eg. i would like to only to allow one strand of DNA be ligated to another DNA molecule to either it's 3'...
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What are common causes of unexpected ligation products?
I digested two plasmids, one with EcoRI and AgeI and the other with EcoRI and XmaI. Digests looked as expected, so I purified the respective fragments and set up the T4 DNA ligation (AgeI and XmaI ...
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How to ligate DNA to an RNA molecule
Is it possible to ligate a small DNA molecule to mRNA? I was reading a protocol from Current Protocols in Molecular Biology and one of the procedures involve ligating a DNA molecule to mRNA using a ...
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