exam #1 - intro molecular lab (cls 607)

preventing contamination in the lab

• Surface decontamination—ethanol in, bleach out

• Nucleases

• Sterile technique

• Reagent handling

• Barrier tips for pipettors

• Separation of pre- and post- amplification areas

basic ingredients in gels for electrophoresis (3)

• Agarose

• Buffer

• Ethidium bromide

how much agarose (%age) should be put in a gel?

• Percentage of agarose used in gel depends on what size of DNA fragments you are interested in

  • Small fragments = larger percentage

  • Large fragments = smaller percentage

pCLS01/lacZ experiment steps

• Isolate plasmid w lacZ gene

• Restriction enzymes to cut specific section of plamid

• Gel electrophoresis to see if the enzymes cut/worked

• PCR of the actual lacZ gene/DNA

• Gel electrophoresis of the lacZ amplicons to see if any DNA was amplified

how to use a nanodrop to find DNA yield and purity

• Nanodrop: spectrophotometer used to find the concentration of DNA, RNA, or protein in a 2 ul drop of sample

• Nucleic acids absorbance max at 260 nm

• For DNA, a 260/280 ratio of 1.8 is considered pure

  • Low 260/280 ratios can indicate contamination with guanidine, phenol, or other reagents used in extraction

DNA quality and the nanodrop

• must compare with DNA ladder to see if the DNA is the one we want

• cannot tell if the product is digested or not (digested DNA absorbs at the same wavelength as undigested DNA)

  • only able to assess QUANTITY & PURITY

restriction enzymes

enzyme isolated from bacteria that cuts DNA at specific sequences

structure of plasmids vs bacterial chromosomes

plasmids are supercoiled ; denatured supercoiled plasmids are like interlocking rings

(plasmid DNA extraction by alkaline lysis) alkaline lysis w P1 buffer

cells are intact & DNA is unaffected by P1 buffer

(plasmid DNA extraction by alkaline lysis) alkaline lysis w P2 buffer

• cells lyse

• dsDNA is denatured to ssDNA

• plasmid ssDNA circles are interlocked, so they remain in close proximity

• chromosome diffuses throughout lysate

(plasmid DNA extraction by alkaline lysis) alkaline lysis w N3 buffer

• 5M K acetate

• plasmids renature in the solution

• insoluble precipitate includes: bacterial chromosome, cell wall fragments, denatured proteins, detergent (SDS)

(plasmid DNA extraction by alkaline lysis) extraction step

• centrifuge tube—bottom will now contain bacterial cell debris (discard); DNA is bound to the column

• plasmid DNA is in the supernatant

• wash to remove excess salt—DNA is still bound to the column

  • column has silica membrane that binds DNA in high salt conditions

• wash again w ethanol to remove salts

• elute w elution buffer—disrupts association between DNA and column

  • plasmid DNA now flows into bottom of the tube

gel electrophoresis

• A technique used to separate DNA fragments by size

• DNA is negatively charged; therefore, it will move toward the positively charged anode (+) when an electric current is applied to the gel

• Gel electrophoresis uses three main ingredients:

  • Agarose

  • Buffer (usually TBE or TAE)

  • Ethidium bromide

(gel electrophoresis) agarose

• When dissolved in buffer and then cooled, it will form a gel with a network of pores

• Small fragments of DNA can move more easily through the pores, so they will move through the gel faster than larger fragments

• Percentage of agarose depends on size of fragments to be separated (usually ranges between 0.5% and 2%)

• Large fragments = use SMALLER percentage

• Small fragments = use LARGER percentage

(gel electrophoresis) buffer

• Main purpose is to carry the charge and maintain the pH during the run

• TBE = Tris-borate-EDTA

• TAE = Tris-acetate-EDTA

• Tris acid keeps DNA deprotonated & soluble

• EDTA chelates magnesium ions which are often cofactors for nucleases

• TAE lowers buffering capacity, TBE has borate which may inhibit downstream enzymes

(gel electrophoresis) ethidium bromide

• Intercalates between base pairs and fluoresces upon exposure to UV light

• Visualization of DNA product

(gel electrophoresis) components of loading the gel

• DNA ladder: solution comprised of different fragments of DNA of known lengths, may already by mixed with a loading buffer

• Loading buffer: has glycerol which helps DNA sink to bottom of well & has tracking dye which helps visualize progression of gel electrophoresis run

what is wrong with the image?

electrophoresis will run off the gel ; DNA migrates in direction of the anode (+)

(gel electrophoresis) voltage for running a gel

• In general, 10 volts for every cm between cathode and anode

• Voltage too high = overheat gel

• Voltage too low = poor resolution

• Run time = depends on size of gel and voltage; keep an eye on tracking dye location to ensure DNA does not run out the end of the gel

polymerase chain reaction (PCR)

laboratory technique used to make a huge number of copies of DNA from a small starting quantity

(PCR ingredients) template & nucleotide mix

• Template DNA: DNA containing the sequence you are trying to amplify

• Nucleotide mix: building blocks of DNA

(PCR ingredients) primers (2)

• Forward primer (5’→3’): short DNA sequence complementary to MINUS strand (3'→5')

• Reverse primer (3’→5’): short DNA sequence complementary to PLUS strand (5'→3')

(PCR ingredients) buffer & Taq polymerase

• Buffer: provides optimal environment for Taq pol

• Taq polymerase: heat stable enzyme that amplifies DNA

PCR steps

• Pre-amplification step if using hot-start Taq

• Amplification cycles:

  1. Denature: time and temp depends on template--usually between 94-98 degrees for 0.5-2 mins

  2. Anneal: temp dependent on primer pair (usually about 5 degrees below Tm) for 0.5-1 mins

  3. Extension: temp depends on what polymerase you are using (usually about 72 degrees) & depends on template and polymerase (usually 1 min/kb)

• Repeat above usually 30 times

what happens if you run too few PCR cycles? too many cycles?

• Too few cycles = not enough product

• Too many cycles = increased chances for nonspecific amplification and errors

considerations when setting up PCR reactions

• Use thin walled 0.2 mL PCR tubes

• Pipette water first

• Dispense reagents into bottom of tube

• Pipette up and down gently to mix, NO VORTEXING

• Centrifuge briefly to ensure all reagents are in the bottom of the tube

preferred blood tubes for molecular testing

• EDTA (lavender)

• ACD (yellow)

human DNA extraction

• Works based on binding of DNA to silica membrane

• First step: lyse cells to release DNA

• Protease K used to break down interfering proteins

• Buffers used to generate correct conditions for optimal binding to column

• Washes help remove additional contaminants

factor V leiden thrombophilia

• inherited blood clotting disorder

• Factor V is converted to Va which interacts with Factor X to convert prothrombin to thrombin

• Activated Protein C inactivates Factor Va

• Mutation of Factor V in codon 506 inhibits the ability of APC to inactivate Factor Va

  • Heterozygous individuals = 7x greater risk of clots

  • Homozygous individuals = 80x greater risk of clots

(FVL) PCR-RFLP

• RFLP: differences in DNA sequences at sites recognized by restriction enzymes

  • Normal individuals: two MnII sites, when you cut their DNA you get three bands

  • FVL homozygotes: missing one MnII site, when you cut their DNA you get two bands

  • Heterozygotes: also three bands but of different sizes than the normal individual

master mix for PCR

mix all the ingredients for multiple PCR reactions (except for DNA) into a tube

• Less pipetting = less chance for error

• Because it is difficult to pipette exactly, typically want to take the amount you need for all your reactions and then add one

general procedure for FVL

• DNA extraction

• PCR of human DNA

• Restriction enzyme digest of FVL PCR product

• Run on gel electrophoresis

• Analyze

normal FVL digest products

3 bands: 180 bp, 81 bp, & 35 bp

FVL homozygote gene products

2 bands: 143 bp & 81 bp

• missing Mnl I restriction site

FVL heterozygote gene products

4 bands: 143 bp, 108 bp, 81 bp & 35 bp