14
votes
Accepted
Restriction enzymes, how are the recognition sequences determined?
This paper describes a simple method to determine restriction sites, which was used to determine the restriction sequence of the previously uncharacterised enzyme from Haemophilus gallinarum.
In ...
- 9,424
12
votes
Accepted
What if target DNA doesn’t have restriction sites
If you're synthesizing the insert then you just design it with restriction sites that are compatible to the vector. Otherwise, you can design PCR primers that have restriction sites in them. That way ...
- 191
6
votes
Accepted
Can restriction enzymes (Type II) displace single-stranded binding proteins (SSB)?
This is an interesting point. In the case of typical restriction enzyme digestion, it is double stranded DNA that is being digested. So in this case the SSBs are presumably binding to the sticky ends ...
5
votes
Why do Type II Restriction Endonucleases cleave at palindromic sequences?
Short Answer
The palindromic symmetry of the restriction site allows a dimeric
enzyme to bind the DNA in a manner that bends the double helix in a
way that facilitates the endonuclease reaction....
- 23.3k
5
votes
Accepted
what's the difference between traditional genetic engineering and and CRISPR?
Bacteria and archaea evolved CRISPR as part of their adaptive immune system to protect themselves from invading viruses and foreign plasmids. The defence system relies on small, non-coding RNA ...
- 225
5
votes
Accepted
DNA Design for Multi-Site Restriction Enzymes
In its active form, SacII (also called Cfr42I) is a homotetramer that must bind two copies of its recognition sequence in order to efficiently hydrolyze its substrate.1 Upon binding two sites on a ...
- 8,206
4
votes
Accepted
Why don't restriction endonucleases digest transformed plasmids?
Laboratory strains used for the purposes of cloning have been genetically engineered to address this issue, typically by deleting genes of the various restriction-modification systems. There are four ...
- 17.6k
4
votes
Accepted
Why is EcoRI supplied with a unique buffer when it is allegedly 100% active in universal buffers?
Activity toward the desired substrate sequence is not the only concern in evaluating a restriction enzyme buffer system. In addition, you need to be concerned about so-called "star activity", or ...
- 1,544
4
votes
Accepted
How to replace intron region in a plasmid?
Your strategy would work, but if possible you might be better off using XmaI rather than SmaI, as the latter is a blunt-end cutting enzyme.
Furthermore, your strategy would not replace the entire gpdA ...
- 628
3
votes
Accepted
Double Digestion with Restriction Enzymes Using Different Buffers
In my experience, a double digestion in CutSmart buffer will work perfectly well. The reaction may proceed slower, but incubate it a little longer and run a gel after the digestion - you'll see ...
- 6,124
3
votes
Double Digestion with Restriction Enzymes Using Different Buffers
If you haven’t already ordered your enzyme, you could get high fidelity KpnI, which has been engineered for use in CutSmart.
You could also consider doing a sequential digest rather than a double ...
- 17.6k
3
votes
Double Digestion with Restriction Enzymes Using Different Buffers
NEB double digest planner is suggesting to use 2.1 buffer for your combination of restriction enzymes (XmaI 50% and KpnI 75% activity). I would be more worried about the star activity of KpnI in ...
- 573
3
votes
Where is cleavage site located?
The cleavage site is always located on the substrate. It is specific sequence of bases (if we speak about DNA or RNA) or amino acids (proteins) that is recognized by the particular enzyme and cut.
- 94
3
votes
Are restriction enzymes active at −20 °C?
Enzymes have temperature optima based on the organism they were isolated from. So I would predict the there is virtually no activity at −20 °C. Another consideration is that the reactions are likely ...
- 3,432
3
votes
Accepted
What happen if we inject restriction enzyme into the blood
The appearance of any foreign antigens (e.g., proteins like restriction enzymes fall n this category) in the circulatory system should trigger an immune response.
There are actually two membrane ...
- 3,432
3
votes
Accepted
Does mung bean nuclease cleave a phosphate group when it's chewing off 5' or 3' ssDNA ends?
According to Kroeker WD, Kowalski W, Laskowski M. 1976. Mung Bean Nuclease I. Terminally Directed Hydrolysis of Native DNA. Biochemistry 15(20):4463-4467
It was concluded that the products of the ...
- 17.6k
2
votes
Accepted
Restriction sites
Most restriction enzymes have a 6bp restriction site (some have 8bp site also). So two restriction sites generally have to span 12bp. However some restriction sites can overlap. For example BamHI and ...
- 35.2k
2
votes
Cleavage of RNA by restriction enzymes?
As to why they don't recognize RNA-DNA heteroduplexes (which are present during transcription, for example), I suspect that the methylation which protects bacterial genomic dsDNA (see the DNA ...
- 3,318
2
votes
Accepted
Cleavage of RNA by restriction enzymes?
This paper found that these enzymes recognize RNA:DNA heteroduplexes. Such duplexes are unlikely to be encountered in vivo. They are present when DNA is primed for replication, but these duplexes are ...
- 17.6k
2
votes
Do restriction enzymes on read 3' to 5'?
Whatever sequence (plus direction) is target, so enzyme will cut. In your example "correct" and "antiparallel" sequences are two different sites.
Actually, your example of 5'-CTTAAG-3' is cut by ...
2
votes
Accepted
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 ...
- 51.1k
2
votes
Accepted
Why doesn’t p53 cause the repair of cellular DNA that has been altered experimentally?
I am no expert in this area, and this answer is only based on a reading of the Wikipedia article on p53, which you should perhaps have read carefully. I welcome edits or correction by persons more ...
- 23.3k
2
votes
What are the possible reasons to get extra unrecognized band in agarose gel electrophoresis?
Looks like genomic DNA contamination to me.
More importantly, does it really matter? I certainly appreciate the curiosity but, if your objective is cloning, I would just keep going and then do some ...
- 17.6k
2
votes
What does the end of DNA look like?
Here is a quick drawing of how BamHI cuts the DNA:
As you can see, after cutting, the two ends are actually the same (always read sequences from 5' to 3').
- 677
2
votes
Accepted
Is chromosomal DNA more likely to interfere with restriction mapping or PCR analysis in E. coli?
Welcome to Biology.SE!
I think you've come to a reasonable conclusion, but not for exactly the right reasons.
The genomes of several strains of Escherichia coli have been sequenced (e.g. https://...
- 5,513
1
vote
Accepted
Do all restriction enzymes have palindromic recognition sites?
The article cited in the question goes on to discuss the different classes of restriction enzyme, for which the recognition ‘style’ differs. Not all recognize palindromic sequences.
Type II ...
- 23.3k
1
vote
Accepted
Cutting dna using restriction endonuclease
Welcome to Biology.SE. I think this question would be better on-topic on math.SE.
Assuming the chromosome is either 4 bases long or infinitely long, then the answer is simply $\left(\frac{1}{4}\...
- 67.8k
1
vote
Why does DNA need to be pure for restriction enzymes to act on it?
Some other macromolecules or other solutes may be present that could interfere with the affinity of the restriction enzyme. For example, there could be salts that denature or modify the conformation ...
- 11
1
vote
Cloning problem. Digestion test at the end of the cloning is always negative
Even minute amounts of undigested vector will contaminate your transformation. You could PCR amplify your insert from the pJet cloning plasmid and then digest the PCR product with BamHI and HindIII. ...
- 695
Only top scored, non community-wiki answers of a minimum length are eligible