MolCellEng_Notes3

MolCellEng_Notes3 - 1 Subcloning 1 Technique to move...

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1. Subcloning 1. Technique to move particular gene of interest from parent vector to destination vector 2. Used for sequencing and protein production 3. 4. Sticky ends anneal 5. Enzyme ligase makes covalent bond b/t vector and fragment 6. Uses recombination instead of restriction sites. 7. Add recombinase Clonase to entry vector with gene & destination vector – moves fragment from 8. Recombinant plasmid gets transformed into bacteria by Chemical Transformation (outer membrane becomes permeable to DNA, add DNA to bacteria, then heat shock) 2. Restriction-enzymes 1. Restriction enzymes in recombinant technology used to cut DNA into many fragments 2. Includes: 1. Hind II (Haemophilus influenza bacteria) GT(T/C)-(A/G)AC 2. Bam HI (Bacillus) G-GATCC 3. Eco RI (E. Coli) G-AATTC 4. Eco RII (E. Coli) –CC(A/T)GG 5. Hind III (Haemophilus) A-AGCTT 3. Gel Electrophoresis 1
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1. DNA fragments separated by size in electric field 2. DNA negatively charged; proportional to size of fragment 3. Separate DNA fragments through gel matrix of agarose or acrylamide 4. Electrophoresis = migration of ions under the influence of an electric field 5. During electrophoresis, F e = F f (net force = zero) 1. F e = electric field force, q = effective charge of ion, E = electric field strength: F e = qE 2. F f = frictional force, f = friction coeff., v ep = electrophoretic velocity
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MolCellEng_Notes3 - 1 Subcloning 1 Technique to move...

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