Midterm Review Feb.06.2015.

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Presentation transcript:

Midterm Review Feb.06.2015

Polymerase Chain Reaction (PCR)

Polymerase Chain Reaction (PCR)

Loading controls for PCR

Polymerase Chain Reaction (PCR) Markers Sample1 Sample2 Band1 ~ 200bp Band2: ~270bp after incorporation of ~70bp sequence Extract the 270bp band for cloning

Polymerase Chain Reaction (PCR) Markers Sample1 Sample2 Band1 ~ 200bp Band2: ~270bp after incorporation of ~70bp sequence Extract the 270bp band for cloning

Cloning

Cloning – Actual E.coli plate

Cloning – Actual E.coli plate

Sanger Sequencing http://www.daviddarling.info/encyclopedia/D/DNA_sequencing.html

Sanger sequencing result Each colored peak corresponds to a single nucleotide at a specific position within the DNA There is one peak per position

Western Blot – Sample Preparation

Western Blot – Gel Electrophoresis

Western Blot - Detection

Western Blot – The actual bands Band intensity corresponds to the amount of protein Antibody against specific protein (here, PKM2) Tubulin protein level used as a control

Western Blot – The actual bands Detection of modified proteins is also possible No difference in ERK protein levels But there is difference between phospho-ERK levels

Fluorescence in situ hybridization (FISH) http://www.semrock.com/fish.aspx -Single or double stranded probes may be used. -Definitely use single stranded probes when targeting RNA. -The probes have to be reverse complementary to the target regions.