5 votes

What is the minimum length required for a circular DNA ligation?

I stumbled across this paper demonstrating it is between 150-200 base pairs. DNA flexibility studied by covalent closure of short fragments into circles D Shore, J Langowski, and R L Baldwin PNAS ...
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  • 309
3 votes

Can someone link me to resources on the efficiency of sticky end ligation?

The best resource for troubleshooting ligations I found (and use frequently) is this NEB page. That is assuming you've already referred to the instructions provided with the enzyme you're using. In ...
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2 votes

Can Bacteria Repair Dephosphorylated Plasmids?

As per WYSIWYG's suggestion, I tried the digest and dephosphorylation again with SalI and ran a gel. The gel showed that SalI was less than ideal at cutting this plasmid. As you can see, the most ...
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  • 5,288
2 votes
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Freezing after restriction digest

From my experience I would say that it is no problem to freeze the digested DNA, when the enzyme is inactivated (even when not, for this time period I wouldn't expect much damage). However, if ...
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  • 49.1k
2 votes
Accepted

circularizing DNA molecules?

For ligation of a linear molecule to occur, the two ends must come together at the active site of the DNA ligase. In a simple molecular cloning experiment the aim is usually to avoid recircularisation ...
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  • 22.6k
1 vote

Separate DNA fragments with very different size (oligo and lambda-DNA)

A spin column might work for your purposes since you can do multiple rounds of binding if your volume is too large to bind in a single pass, but for DNA of that size a lot will be irreversibly stuck ...
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  • 5,203
1 vote

Ligation without purifying insert

Thanks all for your comments guys. I tried purifying using PCR purification kit. I could recover 120 ng of purified fragment from 500 ng. It is ok for my further work.
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  • 630
1 vote

What happens after the purification step in Hi-C sequencing?

So, after the restriction digestion, the sample consists of DNA fragments with 5' overhangs. These are filled in by Klenow DNA polymerase, adding biotin-dCTP to both ends of each fragment. Then the ...
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  • 17.5k
1 vote

Can someone link me to resources on the efficiency of sticky end ligation?

To measure the frequency of indels at the ligation site you can use a vector with a unique restriction site in the lacZ gene. With a colorimetric assay you can count the number of white cfu. Perfect ...
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  • 3,407

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