2. TALEN Kits
TALEN Kits are commercially available and mainly follow two
methods for the construction of TALENs
1) Ligation-independent cloning method- Validated in human cells.
2) Golden Gate cloning method- Validated in multiple organisms,
also optimized for human expression.
3. Golden Gate Kit
Golden Gate cloning is a recently developed method of assembling
multiple DNA fragments in an ordered fashion in a single reaction.
The Golden Gate method uses Type IIS restriction endonucleases,
which cleave outside their recognition sites to create unique 4bp
overhangs (sticky ends).
Cloning is expedited by digesting and ligating in the same reaction
mixture because correct assembly eliminates the enzyme recognition
site.(Engler et al 2009)
TALENs bind and cleave DNA in pairs, and their binding specificity is
determined by customizable arrays of polymorphic amino acid
repeats in the TAL effectors. This collection and accompanying
documentation allow one to efficiently assemble TALEN constructs
with custom repeat arrays, containing anywhere between 12 and 31
repeats. (Cermak et al.,2011)
4. Golden Gate Kit
pZHY500 and pZHY501 are Golden Gate compatible yeast
expression vectors with deletions in the coding sequence that
result in truncations of both the amino and carboxyl portions of the
TAL portion of the protein
pCP5b is an unpublished plasmid that allows one to test TALEN
activity in yeast.
5. Applications:
Human Diagnostics
Detecting viral infections (HIV, etc.)
Detecting bacterial infections (Tuberculosis, etc.)
Genotyping (detecting genetic variants,
which can indicate predisposition to disease)
Scientific Research
Preparing DNA to sequence Monitoring gene
expression levels Manipulating DNA in genetic
engineering and synthetic biology
Thermocycler
6. TALEN electroporation is done with electroporators, appliances that
create an electro-magnetic field in the cell solution. The cell suspension
is pipetted into a glass or plastic cuvette which has two aluminum
electrodes on its sides.
Electroporators
