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
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
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....
- 26.3k
5
votes
Database for commercially available restriction enzymes?
You are probably talking about type II endonucleases, there are a few databases available from the companies which sell these enzymes like NEB or Thermo Fisher. Additionally tools like NEBcutter, ...
- 52.5k
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 ...
- 8,326
4
votes
Accepted
Which was the first restriction endonuclease to be isolated?
The restriction endonuclease (RE) isolated from E. coli K strain was a type I restriction endonuclease, identified/characterized by Meselson and Yuan1 in 1968. Type I REs are site specific, but ...
- 12.9k
4
votes
Accepted
Change of DNA concentration due to restriction digest?
Summary
The poster wishes to know why spectrophotometric readings (presumably absorbance at 260 nm) on a sample of genomic DNA from an unspecified organism appear “slightly” greater after incubation ...
- 26.3k
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 ...
- 638
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 ...
- 17.8k
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 ...
- 1,574
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 ...
- 6,272
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 ...
- 17.8k
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 ...
- 583
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
Linearising plasmids for CRISPR experiment
No. Plasmids should generally be considered as a circular DNA rather than linear. However, no matter where you cut it, the positions don't change.
The position of the cut site is still the position on ...
- 12.9k
3
votes
Change of DNA concentration due to restriction digest?
As you did a clean up, then you lost some DNA in this process. There are always losses from clean up steps.
Note also that spectrophotometry is notoriously unreliable for DNA quantitation; it's more ...
- 12.9k
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://...
- 5,613
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 ...
- 26.3k
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 ...
- 17.8k
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').
- 707
1
vote
Accepted
How to calculate the occurrence of a stretch of nucleotides in a genome?
Strictly speaking the question is from the realm of probability theory. If the probability of a nucleotide $X$ in the genome is $p_X$ ($\sum_x p_X=1$), then the probability to encounter a particular ...
- 3,862
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 ...
- 26.3k
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}\...
- 68.3k
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
1
vote
What can cause incompatible sticky ends to be ligated?
I want to add an extra explanation.
By personal experience I can tell that incompatible sticky-ends can ligate in some conditions. The efficiency will be lower than using compatible ends but still, ...
- 3,128
1
vote
What are the possible reasons to get extra unrecognized band in agarose gel electrophoresis?
Could be several things...just off the top of my head. Without your vector map and expected sizes it will be almost impossible to say for sure.
You'll probably also have to give us more information ...
- 1,301
1
vote
Plasmid specific DNA degradation
traditional method to remove a plasmid from E. Coli is to grow the cells with Etidium bromide.
http://vlab.amrita.edu/?sub=3&brch=186&sim=1097&cnt=1
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Only top scored, non community-wiki answers of a minimum length are eligible