“A” LB/amp =100µl/1000µl= 1/10= .10µl was the volume of the fraction of cell suspension spread.
MASS OF PLASMID SPREAD ONTO “A” LB/amp PLATE. FORMULA.
MASS PLASMID SPREAD
= total mass plasmid x fraction spread
.05µg of pAMP (and pKAN) was the total mass.
.10µl was the volume of the fraction of cell suspension spread.
.05µg * 0.10µl = .005 µg/µl was the amount of the mass plasmid spread per pAMP (and pKAN).
NUMBER OF COLONIES PER µg OF pAMP & pKAN. WHAT DO YOU USE?
TRANSFORMATION EFFICIENCY =
colonies observed
-----------------------
mass plasmid spread
pAMP =4,212 CFU/.005 µg = 8.424 X 105 colonies/ µg.
HOW DOES ONE PREPAIR COMPETENT CELLS?
START WITH MID LOG
SPIN TO PELLET
SEASON WITH CaCl ICE BATH FOR 20
SPIN AND DRAIN
SEASON WITH CaCl
VORTEX AND STORE.
HOW DOES ONE TRANSFORM E. COLI WITH RECOMBANT PAMP/PKAN PLASMIDS?
COMPENTENT CELL TO +PAMP THEN ICE
ADD LIGATED TO SAME TUBE THEN ICE 20
HEAT SHOCK 42 THEN ICE
LB TO TUBE (ALL) 37 FOR 50
SPREAD ON PLATES
INCUBATE 37 12-24HRS.
THE SOUTHERN BOLT TECH RELIES ON WHAT?
HYDROGEN BONDSTHAT HOLD TOGTHER COMPLEMENTARY BASE PAIRS IN DOUBLE STRANDED DNA.
AT TEMPS ABOVE ---- OR pH ABOVE ----- WHAT HAPPENS TO HYDROGEN BONDS?
ABOVE 90
pH 10.5
HYDROGEN BONDS ARE DISRUPTED AND COMPLEMENTARY STRANDS DENATURE INTO SINGLE STRANDS.
3 SOURCES OF SINGLE STRANDED DNA HYBRIDIZATION PROBES.
RESTRICTION FRAGMENT
PLASMID CONTAING CLONED SEQUENCE
SYNTHETIC OLIGONUCLEOTIDE
NAME 3 WAYS TO VISUALIZE A PROBE
RADIOACTIVE
COLORMETRIC
CHEMILUMINESCENT
NAME THE BASIC STEPS OF THE S BLOT
RESTRICTION -->GET FRAGMENTS
RUN GEL OF LAMDA DNA
STAIN GEL, RINE, UV VIEW &PHOTO
DENATURE (DOUBLE RINSE)
BLOT
WASH & BAKE NYLON MEMBRANE
PREHYBRIDZE & HYBRIDIZE
WASH & DEVELOP
AT THE BEGINING OF A TYPICAL SOUTHERN HYBRIDIZATION-------- OF GENOMIC DNA IS CUT WITH ONE OR MORE ------- -------.
10ug
RESTRICTION ENZYMES
A --------- WITH A ----BP RECOGNITION SEQUENCE STATICALLY CUTS ONCE EVERY ----- BP(X).
ENDONUCLEASE
6
4,096 (46)
E.COLI HAS HOW MANY BP IN GENOME AND HOW MANY RESTRICTION FRAGMENTS?
4 MILLION BP
977 RESTRICTION FRAGMENTS
IN SOUTHERN BLOTTING YOU REALLY DON'T GET SMEARS. WHY?
SMALL SIZE OF LAMDA GENOME (48,502) AND THE LARGE DNA LOAD (6ug) ENSURE REPRODUCIBLE RESULTS AND SHORT HYBRIDIZATION TIMES.
SOUTHERN BLOTTING IS SO SENSITIVE THAT.....
ITS CAPABLE OF DETECTINGH A RESTRICTION FRAGMENT THAT IS PRESENT ONLY ONCE IN THE HUMAN GENOME.
HOW MANY BP SND RESTRICTION FRAGMENTS IN HUMAN GENOME?
3.5 BILLION
854,492 FRAGMENTS
THREE METHODS ARE COMMONLY USED TO TRANSFER NUCLEIC ACIDS FROM GELS TO NYLON NAME THEM.
1. CAPILLARY..SOUTHERN'S IDEA
2. ELECTROPHORETIC..2-3 HRS USING CURRENT, BUT BUFFER MUST BE CYCLED TO BE COOL. BEST FOR POLYACRYLAMIDE.
3. VACUUM FASTEST. MOST EFFICENT. VACUUM FROM THE BOTTOM.
it’s flammable. Nucleic acids are draw to it via hydrophobic interactions. WHO AM I?
Nitrocellulose MEMBRANE
WHAT MEMBRANE BINDING IS REVESABLE AND WHAT ABOUT THE OTHER?
NITROCELLULOSE.
NYLON MEMBRANES BIND NUCLIC ACIDS IRREVESTABLE UNDER LOW IONIC STRENGTH.
NYLON CAN UNDGO MANY HYBRIDIZATION WHERE AS NITOCELLULOSE CAN'T.
TRUE?
TRUE.
THE SOLE DISADVANTAGE OF NYLON MEMEBRANE IS...
SOMETIMES GIVE HIGHER LEVELS OF BACKGROUND, ESPICALLY WITH RNA PROBES.
WHAT FILTER PAPER SPEC IS REFERENCED IN THE TEXT?
WHATMAN 3mm
P = PAPER TOWELS (FILTER IS BENEATH)
A = AGAROSE GEL
W =WICK
N = NYLON MEMBRANE
S = HIGH SALT SOLUTION
WHAT TYPE OF BUFFER IS PLACE IN THE TRAY?
500 ml OF 10x SSC BUFFER
WHEN YOU WASH AND BAKE AFTER BLOTTING WHAT IS THE SOLUTION THATS USED?
WASH WITH 100ml OF 2X SSC BUFFER FOR ONE MINUTE, THEN BAKE AT 70 FOR 30MNS OR LONGER.
DENATURE AND NEUTRALIZE DNA STEP...WHAT IS IT?
STEP BEFORE YOU BLOT.
DENATURE FOR 15MNS TWICE
THEN
NEUTRALIZE FOR 15MNS TWICE
NAME THE STEPS AFTER THE BLOTTING
WASH & BAKE
PREHYBRIDIZE & HYBRIDIZE
PROBE
HYBRIDIZATION TO EURKARYOTIC GENMOES ARE USUALLY CARRIED OUT AT ---- IN AQUEOUS SOLUTIONS OR AT ----- IN -----------.
68
42
50% FORMAMIDE
HYBRIDIZATION AT LOWER TEMPS IN FORMAMIDE IS LESS DAMAGING TO THE NITROCELLULOSE MEMBRANE BUT......
TAKES 2-3 TIMES LONGER THAN AT HIGHER TEMPS IN AQUIOUS SOLUTION.
THE RATE OF DNA REASSOCIATIONG INCREASES AS THE THE VOLUME--------
DECREASES.
THUS REDUCING THE AMOUNT OF PROBE REQUIRED AND HYBRIDIZIATION TIME.
T OR F?
THE USE OF A BLOCKING REAGENT IS SOMETIMES OMITTED HEN USING NYLON MEMBRANES BECAUSE THE PROTEIN CAN INTERFER WITH PROBE BINDING.
TRUE
NON FAT MILK OR BOVINE SERIUM ALBIUMIS USED FOR WHAT IN BLOTTING?
BLOCKING REAGENT
NAME 3 TYPES OF PROBE DETECTION
RADIOIACTIVE...OLD AND DANGEROUS
COLORIMETRIC
CHEMLLUMINATION
WHAT PROBE USES DIGOXIGENIN OR BIOTIN?
COLORIMETRIC
WHAT PROBE TYPE USES FILM AND A DARKROOM?
CHEMILUMINSCENTATION
T OR F?
WHEN USING RESTRICTION FRAGMENTS AS THE PROBE, YOU SHOULD INCREASE THE STRINGENCY OF THE HYBRIZIDZATION
NAME THE DETAILS.
TRUE
65 AND 5X SSC BUFFER
THIS PLANT STEROID MOLECULE IS HIGHLY ANTIGENTIC AND IS USED AS A PROBE FOR IMMUNE DETECTION.
DIGOXIGENIN
WHAT PLANTS DOES THE DIGOZIGENIN COME FROM?
FOXGLOVE & DIGITALIS
HOW DOES ONE PREPAIR DIGIOZIGENIN?
IMMUNE SHEEP
GET IGg VIA ION EXCAHANGE CHROM.
ISLOATE VIA IMMUNOSORPTION (COLUMN)
LINKED TO ENZYME ALKALINE PHOSPHATE.
YOU HAVE Ab/ENZYME PROBE THAT FORM IMMUNE COMPLEX (PROBE).
ALKALINE PHOSPHATE CATALYZES THE REMOVAL OF A ----- GROUP FROM
5-BROMO-4 CHLOR-3 INDOLPHOSPHATE
(X PHOSPHATE). THE RESULTING OXIDATION DIMERIZATION FORMS WHAT?
PHOSPHATE
INDGO PARCIPATANT
HYDRIDE IONS RELEASED DURING DIMERIZATION REDUCE A SECOND COMPOUND. WHAT?
NITRO BLUE TETRAZOLIUM (NBT) WHICH FORMS A PUPRPLE PRECIPITATE.
