Plasmids experiments

Plasmid prep
Resuspension solution
50mM glucose: increase solute conc outside bacteria, draw water and plasmid out when membranes are destabilised
25mM Tris-HCl pH8.0
10mM EDTA: binds Ca2+ (membrane stabiliser) and Mg2+ (cofactor of DNase)
50ug/ml RNase A

Lysis solution
0.2M NaOH: denatures DNA into single strands, but circular plasmid remain interlocked
1% SDS: dissolves cell membrane

Neutralisation solution
3M K acetate pH5.5: KDS is insoluble, ppt lipid, protein and chromosomal DNA, plasmid DNA renatures with neutralisation

Alcohol precipitation
0.7x vol isopropanol or 2x vol ethanol: forms H-bonds with water, polarisablility of alcohols much less than water
monovalent cation: neutralises charge on DNA, permitting DNA to aggregate

A260= 1.0 for 50ug/ml double stranded DNA
A260/A280> 1.6 (protein contamination reduces ratio)

Restriction Digest
1unit of enzyme is defined as the amount required to digest 1 ug of phage lamba DNA in 1h in a volume of 50ul. Usually 1-5u used in 20ul (4ug of plasmid DNA).

Agarose Gel Electrophoresis buffer
Tris-acetate-EDTA (TAE) or : better resolution, preferred for gel purification
Tris-borate-EDTA (TBE): better buffering capacity
EDTA chelates Mg2+ (cofactor of DNase)

EtBr/ SybrSafe: slip in between the stacked base pairs of double-stranded DNA

Transformation
Making competent cells
Resuspend pellet in 1/2 vol of 0.1M cold CaCl2. Incubate on ice.
Resuspend in 1/10 vol of 0.1M cold CaCl2.

Transformation
Incubate with plasmid on ice for 1h
Heat shock
Grow out
Plate
Typical transformation, use 0.01-0.1ug DNA, if from cloning (not supercoiled), need more
Typical transformation frequency: 10^6 Tsf/ug DNA
Typical TSF for electroporation: 10^10TSF/ug DNA