MolCellEng_Notes3 - 1 Subcloning 1 Technique to move...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
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
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
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
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

Page1 / 7

MolCellEng_Notes3 - 1 Subcloning 1 Technique to move...

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online