F13 7 Microbial classification(1)

Density gradient various allows differentiation you

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Unformatted text preview: ENSITY GRADIENT (VARIOUS) ALLOWS DIFFERENTIATION YOU CAN MAKE CONCENTRATION GRADIENT WHERE YOU HAVE LOW DENSITY AT THE TOP AND HIGH DENSITY AT THE BOTTOM. IN OTHER WORDS, TOP AND BOTTOM DIFFER IN VISCOSITY IF YOU TAKE DNA FROM 2 DIFF ORG. AND MIX WITH ETHIDIUM BROMIDE+CENTRIFUGE, IF THERE ARE DIFFERENCES IN DNA MOLECULES THAT CAN BE DETECTED, ALL YOU HAVE TO DO IS SHINE A LIGHT TO CONFIRM DIFF IN ORG DNA MELTING TEMP: IF YOU HAVE A STRAND OF DNA AND THEY;RE MATCHED, THE H BONDS HOLD THEM TOGETHER. PHOSPHO RIBOSE STRANDS HELD BY STRONG COVALENT BONDS. MORE GC BONDS=HIGHER TEMP NEEDED BEFORE STRANDS COME APART REASON BECAUSE THE 3 H BONDS V. 2 AT H BONDS; MORE AT = WEAK BONDS HEAT GENTLY WILL CAUSE THE H BONDS TO BREAK AND STRANDS WILL SPLIT ORDER OF BASES WILL REMAIN INTACT EVEN AFTER STRANDS COME APART IF YOU COOL THEM SLOWLY, THEY WILL REANNEAL, SINCE THEY ARE ANTIPARALLEL AND COMPLEMENTARY, THEY WILL NATCH UP TO OLD STRAND AND FORM AN INTACT MOLECULE. DNA HYBRIDIZATION: (23MINS), still used; YOU CAN CLONE THE ORGANISM YOU WANT TO DETECT AND PUT A SEQUENCE INTO AN E COLI, CAN USE TO DETERMINE UNKNOWN COMPOSITION "IS THERE SALMONELLA ON PLATE?"; DETERMINES RELATION BUT NO EXACT RELATIONS; GOOD WAY AT LOOKING AT RELATEDNESS, LED TO ABILITY TO SEQUENCE RIBOSOMES RIBOSOME SEQUENCING: USED TO COMPARE BECAUSE EVERY CELL HAS RIBOSOMES; 16S WAS CHOSEN; BASICALLY, THE 16S IS MADE OF 1500 BASE PAIRS Template=>chromosome of unknown (contains 16s rDNA) PROBES: DEFINE 16S RIBOSOM GENE, NOTHING ELSE Primers=>Conserved, bookend 16s rDNA DNA polymerase=>enzyme dNTPs=> (ATP, GTP, CTP & TTP) Thermal cycle=>melt->anneal->extend MONOMERS: BASIC NUCELOTIDES PRIMER IS SPECIFIC TO 16S ~1,500 base pairs DENATURE + ANNEAL + EXTEND ORIGINAL TEMPLATE STRAND DELUDES OUT, END UP WITH SEQUENCE OF INTEREST. 1 MILLION COPIES MADE AFTER 25 CYCLES. POLYMERASE CHAIN RXN: TAKE DNA STRAND-DENATURE AT 94-96 CELCIUS,, CAUSE STRAND TO COME APART AND THEN THESE PROBES WILL BIND AND THE IDEA IS THAT YOU WILL FIND AND DEFINE THE 1...
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This document was uploaded on 02/25/2014 for the course MICROBIOLO 101 at San Jose State.

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