Plasmids and their uses Flashcards
What is the detection limit for dsDNA in gel electrophoresis ?
A single band at 10ng.
What are the consequences of DNA overloading ?
Railing and smearing, more severe with longer fragments
What are the three main purposes of the gel loading buffer ?
- Increase density (sucrose, glycerol, Ficoll) of sample (=>evenly distributed in well)
- Aid loading by adding colour
- Add mobility dye(s) Bromphenol Blue/Xylene cyanol FF/Orange Green to monitor electrophoretic process
Why are the smaller fragments harder to identify ?
Smaller fragment = smaller region to which the DNA-binding dye attaches to –> less intense signal under UV light
What is the structure and function of alkaline phospohatase Calf Intestinal (CIP) ?
AP = = enzyme that dephosphorylates 5’ and 3’ end of DNA, RNA, dNTPs and proteins –> prevents self-ligation of plasmids
Dimeric metalloenzyme of two identical subunits, each subunit contains two zinc ions and one magnesium ion
What is the optimal pH of AP ?
An alkaline solution is optimal, with a pH optimum at 8 - 10 but can be used at reduced activity at (pH = 7.5-9).
1 unit of CIP will dephosphorylate ~ 1 pmole of 5’ phosphorylated termini at 37 ̊C within 30 minutes.
You have 1 μg of a 3 kb plasmid and the CIP has a concentration of 0.2 U/μl.
How much enzyme do need to add?
Average MW of 1bp ~ 650 Daltons = 650 g/mol
MW of our 3 kb fragment = 3,000 * 650 = 1.95e6
Number of moles : (1e-6) / (1.95e6) = 5.13e-13 moles = 0.513 pmol
Number of units needed: 0.513 (1 U for 1 pmol)
0.513 / 0.2 = 2.565 uL of enzyme
Because we have 2 DNA strands, we need 2.565 * 2 = 5.13 uL of enzyme
What is Southern blotting ?
Northern blotting ?
Western blotting ?
Southern blotting (named after Sir Edwin Southern) = identification of DNA Northern blotting = identification of RNA Western blotting = identification of proteins
What are the three main gel blotting techniques ?
What are the advantages and limits of each ?
Capillary blotting: - transfer time = 2-16hrs - membrane = nitrocellulose or nylon - equipment = standard - resolution = excellent (short transfer) Vacuum blotting: - transfer time = 0.5-1h - membrane = nylon - equipment = special - resolution = excellent (optimal vacuum) Electrophoretic blotting: - transfer time = 2-16h - membrane = nylon - equipment = special - resolution = good (external cooling)
What is the size limit of inserts accepted by plasmids ?
How does this affect their use ?
<10kb (nat occurring multi-copy plasmids)
Used for sub-cloning and downstream manipulation, cDNA cloning and expression assays
What is the size limit of insert accepted by phages ?
How does this affect their use ?
5-20kb (e.g. bacteriophage lambda)
Used for genomic DNA cloning, cDNA cloning and expression libraries
What is the size limit of insert accepted by cosmids ?
How does this affect their use ?
35-45kb (e.g. plasmid containing a bacteriophage lambda cos site)
Used for genomic library constructions.
What is the size limit of insert accepted by BACs ?
How does this affect their use ?
75-300kb (e.g. E. Coli F factor plasmid)
Used for the analysis of large genomes.
What is the size limit of insert accepted by YACs ?
How does this affect their use ?
100-1000kb (e.g. S. Cerevisiae centromere, telomere, and autonomously replicating sequence)
Used for analysis of large genomes, construction of YAC transgenic mice.
What is the size limit of insert accepted by MACs ?
How does this affect their use ?
100kb to > 1 MB (e.g. mammalian centromere, telomere and origin of replication)
Under development for use in animal biotechnology and human gene therapy.
What are the cos ends of the lambda genome ?
How are they produced ?
What are their properties ?
cos ends = 12 nucleotide 5’ overhangs of linear DNA are the result of a cut by the enzyme terminase, which is encoded by itself and acts like a restriction enzyme during the replication of the phage DNA. The terminase is an endonuclease specific for the cos-site in multimeric phage DNA. The ends of the resulting monomeric DNA are similar to cohesive ends produced by common restriction enzymes. Because they have long 5’ overhangs cos ends are much more sticky than the cohesive ends generated by restriction enzymes.
cos end : 5’ - G/GGGCGGCGACCTC - 3’
What is the function of DNA ligase ?
Joins 5’-phosphate to 3’-OH (phosphodiester bond) within
double stranded DNA using Mg2+ and ATP or NAD+.
What is the difference between E. Coli DNA ligase and T4 DNA ligase ?
E. Coli DNA ligase : uses NAD+ as a cofactor, joins only cohesive ends
T4 DNA ligase : uses ATP as a cofactor, joins cohesive and blunt ends of DNA, oligonucleotides, RNA and RNA-DNA hybrids
What are the four steps of the ligation reaction ?
Step 1 : DNA ends have to collide by chance and stay together long enough for the ligase to join them => easier at low T ̊C.
Step 2 : Self-adenylylation of an active site lysine using ATP or NAD+ (depending on the ligase) release of pyrophosphate (PPi)
Step 3 : Transfer of the adenylyl group to the 5’-phosphorylated terminus (donor) forming apyrophosphate bond.
Step 4 : Nucleophilic attack of 3-hydroxyl group (acceptor) on 5’- phosphate group (donor), loosing the AMP, forms
phosphodiester bond.
Are sticky ends produced by the same restriction enzyme compatible.
What about ends formed by different restriction enzymes ?
Same enzyme = frequently yes
Different enzyme = sometimes, e.g. BamHI and Sau3A, PstI and Nsil
