Biotech Test

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Vector Cloning:

1. Isolate gene of interest
2. Cut using restriction enzymes (2 cut sites)
3. Take plasmid from bacteria
4. Cut using the SAME restriction enzymes (1 cut site)
5. Put gene of interest into plasmid using the sticky ends, glued together by ligase
6. RECOMBINANT DNA/ TRANSFORMATION
7. Culture
8. "Infect"

How to isolate the gene of interest: gel electrophoresis

Fragments get cut at various lengths.  Moves from NEGATIVE --> POSITIVE

Complete digestion: all fragments cut at restriction site

Incomplete digestion: all possible fragments are cut

VECTOR CLONING EXPRESSES THE GENE

Agarose Gel Electrophoresis Video

PCR WILL NOT

Polymerase Chain Reaction:

1. Denature DNA, break hydrogen bonds
2. DNA primers, dNTPs & Taq polymerase
3. Repeat

Replication is EXPONENTIAL after the THIRD CYCLE

Restriction Fragment Length Polymorphism

R -restriction enzymes

F- complete digestion

L -PCR

P- patterns (comparison)

DNA Sequencing (Sanger Method)

1. Denature DNA, break hydrogen bonds
2. DNA primers, dNTPs, ddNTPs & Taq polymerase
3. ddNTPs stop replication
4. PCR
5. Complementary sequence read from bottom to top

http://www.dnalc.org/resources/animations/sangerseq.html