Tutorial 1 Flashcards
5’ end overhang
5’ A 3’
3’ TCCGTATT5’
5’ end recessed
5’ AGGCATAA 3’
3’ T 5’
3’ end overhang
5’ AGGCATAA 3’
3’ T 5’
3’ end recessed
5’ A 3’
3’ TCCGTATT5’
What is the efficiency patter of T4 PNK over different 5’ ends?
5′ overhangs efficiently, follow by blunt-ended and, then 5′ recessed ends. The two last ones are associated with a reduced efficiency
Blunt end
5’ AGGC ATAA 3’
3’ TCCG TATT5’
Canonical
Stick to a canon, what is usually expected to see.
For example DNA and RNA base pairing sticking to Watson-Crick rules of hybridization
Non- Canonical
Does not stick to a canon, what is unexpected to see.
For example non-canonical base pairing occurs when nucleobases hydrogen bond, or base pair, to one another in schemes other than the standard Watson-Crick base pairs
Why are OriR’s high in T and A?
A-T base pairs are held together with two hydrogen bonds not three as G-C pairs are. As a result, stretches of DNA that are rich in A-T pairs can be separated more readily at lower temperatures and allows the replication machinery room to come in and get busy making copies.
What does it mean we have a “relax” Ori?
- Positively regulated by RNA or proteins
- No extra machinery needed
- High yield as a result
What does it mean we have a “stringent” Ori?
- Negatively regulated by RNA or proteins
- Extra machinery from the host is needed
- Low yield as a result
Are plasmid with the same original compatible or incompatible?
Incompatible because they will compete for the same machinery, creating an unstable and unpredictable environment
What genes encodes for the Methyltransferases for Adenine and Cytosine?
The dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes that encode for the Dam and Dcm methyltransferases.
What bases can suffer a methylation process?
Cytosine and Adenine
Why is methylation important when doing enzyme digests?
Methylation should be considered when digesting DNA with restriction endonucleases because cleavage can be blocked or impaired when a particular base in the recognition site is methylated
Solution to undigested methylated restriction sites when cleaving DNA in the lab
Restriction sites that are blocked by Dam or Dcm methylation can be un-methylated by cloning your DNA into a dam–, dcm– strain of E. coli, such as dam–/dcm– Competent E. coli
What is a MCS?
A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites - a standard feature of engineered plasmids.
What is the purpose of MCS?
The purpose of a MCS in a plasmid is to allow a piece of DNA to be inserted into that region.
What are MCS also known as?
Polylinkers
Star activity
is the relaxation or alteration of the specificity of a restriction enzyme mediated cleavage of DNA. The result is typically cleavage at non-canonigal recognition sites (which are recognition sites that differ from the normal, expected one), or sometimes complete loss of specificity.
What substances can inhibit a reaction ( substrates)
Ethanol
Why adding to much enzyme can inhibit a reaction
The problem is not the amount of enzyme (although in massive quantities it can induce a start activity), but mainly with the glycerol, as all enzymes are stored in this substance and using more than the advice quantity can inhibite the reaction.
