Question

Accurately describe the two restriction enzymes (EcoRI and HindIII). Both restriction enzymes were used for Gel Electrophoresis.

Answer 1

EcoRI :

Clevage sites: 5-GAATTC-3

3-CTTAAG-5

Source: Escherichia coli RY13

Concentration: 8-20 units/ul

Supplied buffer: H

Reaction temperature: 37°C

Substrate : lambda DNA

Effect of DNA methylation: Enzyme activity is affected by CG methylase depending on the sequence following the recognition site.

Activity: Unrelated site may often be cut in the presence of high concentration of glycerol, Mn²⁺ and at low ionic strength. Enzyme activity reduces to 80% by the addition of spermine (ca. 0.2mM), but star activity reduces to 50%.

HINDIII :

Clevage Sites : 5-AAGCTT-3

3-TTCGAA-5

Source :  Haemophilius influenzea Rd

Concentration : 10-20 units/ul

Supplied Buffer : M

Reaction temparature : 35 degree c

Substrate : Lamda DNA

Activity : Unrealted site cut in the presesnce of DMSO or mn++

ECORI AND HINDIII USES FOR GEL ELECTROPHORESIS :

A restriction enzyme will search for a specific sequence of base pairs. It will cut a piece of DNA at that specific arrangement of base pairs. When different restriction enzymes are used to cut a single strand of DNA such as the Lambda DNA, fragments of varying sizes are produced. These fragments can be separated by their size through gel electrophoresis.The restriction enzymes used in this investigation are EcoR1,HindIII and use gel electrophoresis to separate the resulting DNA pieces. To help see the pieces, stain the gel with a chemical that will combine with the DNA causing it to take on a blue colorand and it helps to identify restriction enzymes and their specificity. It determines the number of restriction sites on Lambda DNA.