Lecture One Ctd. - Cloning
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What are some purposes of DNA Methylation?
- DNA protection from phage infection.
- Gene Expression.
- Regulation of DNA replication.
- Mismatch Repair.
- Recombination - Incorporating new fragments.
Are all DNA Methylases involved in Restriction/Modification systems?
No, some act seperately.
What are some examples of Methylases in E.Coli that are not part of a Restriction/Methylation system?
- Dam Methylase
- Dcm Methylase
- EcoKI Methylase
How does Dam Methylase work?
Dam Methylase Transfers a Methyl Group from s-AdenosylMethionine (SAM) to a specific Adenine.
What is the specific sequence that Dam Methylase acts on?
GATC - The A is Methylated at it's N6 position.
What sequence(s) does Dcm Methylase act on?
What is an Approximate of recognition sites for Dcm Methylase?
What is the frequency of occurrence of sites recognized by EcoKI Methylase?
~ Once every 8 Kb
Which Non-Restriction/Modification Methylase has the rarest recognition sites?
What position on Cytosine does Dcm Methylase Methylate?
The C5 position.
If a Methylase has a recognition sites that frequently occur, what impact does this have on Methylation of the DNA?
There will be a higher frequency of Methylation/More DNA will be Methylated.
What is MboI?
A Restriction Enzyme Cleaving at GATC sites.
Does Methylation of DNA improve or inhibit transformation of Recombinant E.Coli?
It will inhibit it, because bulky Methyl groups block Nuclease binding during Transformation.
Name a Eukaryote Methylase.
How does CpG Methylase work?
CpG Methylase transfers a Methyl Group to the C5 position on cytosine.
What are some characteristics of Methylation in Eukaryotes?
Eukaryote DNA is highly Methylated. What effect would this have on it's digestion?
It would be harder to digest with most Restriction Enzymes
Is Methylated DNA always protected?
No, some Restriction/Modification systems specifically target Methylated DNA.
Mcr and Mrr are which type of Restriction/Modification systems?
Type IV (They require Methylation)
What is a something to be careful of when using Type IV Restriction/Modification systems?
That these systems do not digest Plant and Animal DNA as they are heavily Methylated.
When might mixing Mcr/Mrr (from bacteria) Systems and Eukaryote DNA be an issue?
When transforming bacteria with Eukaryote DNA in cloning experiments.
What are Isochizomers?
Enzymes isolated from different organisms that cut the same sequence in the same place (albeit under different conditions).
What are Neoschizomers?
Enzymes that cut at the same recognition site, with a different point of cleavage.
K12 is another name for?
Wild Type E.coli
AntI is a Restriction Enzyme with the Recognition Sequence;
If you wish to digest Methylated DNA at this sequence, how might you do this considering AntI cannot affect Methylated sites?
You could find an Isochimer of AntI that will cleave Methylated sites.
HpaII is a Restriction Endonuclease acting on Cytosine. Will it cut Methylated or UnMethylated Cytosine?
MspI is a Restriction Endonuclease acting on Cytosine. Will it cut Methylated or UnMethylated Cytosine?
What does NEB stand for?
New England Bioloabs
What are the three main NEB buffers called?
What features are found in all NEB buffers?
What are the unique constituents of NEB 1.1?
- 10mM Bis-Tris-Propane HCl
- pH 7.0 @ 25°C
What are some unique features of NEB 2.1?
- 50mM NaCl
- 10 mM Tris-HCl
- pH 7.9 @ 25°C
What are some unique features of NEB 3.1?
- 100mM NaCl
- 50mM Tris HCl
- pH 7.9 @ 25°C
What does BSA stand for?
Bovine Serum Albumin.
Is BSA necessary for all Enzymes? If not, why is it present?
BSA is a useful constituent in most enzymes but not all. It is left in buffers because if it is not used, it does not hinder Enzyme Activity.
What effects does BSA have to increase efficiency?
Lessens Enzyme loss on tips and tubes.
Why is Mg2+ present in most buffers?
It is a Cofactor for many of the DNA interacting enzymes.
What is a crucial factor in buffers? (Key difference between them).
Does Ionic Strength affect Enzyme Activity?
Yes. It is essential.
Some older buffers use detergent. What detergent do they use?
What is the Unit of measurement for Enzymes?
Just called Units. It is the amount of Enzyme that will completely cut all sites on 1μg of DNA in one hour in a 50μL reaction volume.
What are the most common DNA's used to measure Enzyme activity?
Lambda or pBR322 DNA.
Why are Enzymes not measured by Mass or Volume etc.
Because not all Enzymes in an Enzymatic Solution may still be active.
What are two factors affecting DNA quality (digestibility)?
- Contamination with RNA
- Contamination with Protien
What is Star activity?
Altered Enzyme Activity whereby Enzymes cut at sites different from their recognition sites.
What factors can result in Star Activity?
- >5% Glycerol
- <100 units of Enzyme/μg of DNA
- Prolonged Reaction Time
- Presence of Organic Solvents
- Non-Optimal Buffer
What effects can high Glycerol have on Restriction Enzyme activity?
What effects can the presence of Organic Solvents have on Restriction Enzyme Activity?
What are some things to consider when storing Enzymes?
- They are temperature sensitive (non-frost-free freezer)
- They must be stored in glycerol
What are some things to consider when handling enzymes?
- Do not overpippet (there'll be too much glycerol)
- Add last
What is an Example of a control when using enzymes?
Digest a known number of sites on DNA with the enzyme you are using to confirm activity.
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