PCR has how many cycles
25-40
1st step in PCR cycle
Denaturation
Two strands will separate
Denaturation
2nd step in PCR cycle
Annealing
forward primer will bind complementarily on the first few strands on one strand
reverse primer will bind on the last few strands of the target DNA on the other strand
Annealing
Third step in PCR cycle
Elongation
enzyme responsible for strand to elongate, with the help of magnesium
DNA polymerase
Product of PCR
AMPLICONS
Denaturation temp
94-95
Primer annealing
55-65
Extension of DNA temp
72
Number of cycles in PCR review
25-40
Many copies of DNA molecule to be sequenced
Madam Gilbert method
contain a chemical that cuts after Guanine
G tube
Still need many copies of DNA
Sanger megthod
ddGTP – black o ddCTP – blue o ddATP – green o ddTTP - red
Basis of automated sequencing
- Based on single tube sanger sequencing
- Has color coded nitrogenous bases
- DNA fragments with different color codes are read by
a laser, then resulting sequence will be flashed on the
machine screen - Each color band represents a particular nitrogenous bases
Automated sequencing
You have to blot the specimen on a filter paper or nitrocellulose membrane
Dot blot
because the DNA or the strand of
target organism is being sandwiched by two nucleic acid probe (unlabeled capture probe and signal generating probe or labelled probe.)
Sandwich
Most commonly used label in hybridization. Kit
Streptavidin phycoerythritin conjugate
Probe can be detected with a fluorescent antibody in a technique called f
Flouresecence in situ hybridization
Used to denature proteins to further purify
the sample. To remove proteins to have a
pure DNA suspension
Sodiumdodecyl. Sulfate
better to be used in DNA
electrophoresis agarose gel
Polyacrylamide gel
