1. Today  Housekeeping  Gel electrophoresis  (Complete DNA extractions)  Prepare an 1% (w/v) agarose gel  Run DNA on the gel (45 min)  Spectrophotometry

2. http://www.biotechniques.com/multimedia/archive/00250/BTN_A_000114307_O_250227a.pdf

3. 15 minute powerpoint topics Discovery of DNA structure Restriction enzymes Southern blotting Cloning The first sequenced gene PCR, specificity and sensitivity q-PCR BAC libraries ESTs BLAST and database searches Microarrays Forensics (Genome sequencing, the $1000 genome) Next generation sequencing Bioinformatics Epigenetics "non-coding" RNA C-value paradox Phylogenetic genomics Archeological genomics

4. PARASITES AND SNAIL BIOLOGY “identity, possibilities” phylogenetics “intentions” transcriptomics PCR rDNA/mito Bioanalyzer DNA-free, direct sequencing gel electrophoresis nanodrop spec Sequence ID (BLAST) editing Phylogenetics electrophoresis RT-PCR gel CTAB, DNAzol Trizol TA cloning, B/W screening M13 sequencing Primer design, walking Qiagen plasmid extraction Restriction digests DNA RNA GenBank submission

6. DNA extracted? How much and what quality? Concentration, Amount, Contamination, Degradation Good to know for downstream applications several assays available START POURING GELS (steps 1-9 handout) Read, THEN do, check off progress

7. Horizontal agarose gel electrophoresis “Jello”, 1% agarose in TAE SIDE VIEW TOP VIEW

8. Horizontal agarose gel electrophoresis Apply DNA in “loading dye buffer”

9. Horizontal agarose gel electrophoresis DNA Apply voltage short gel vs long gel

10. Horizontal agarose gel electrophoresis DNA Pulling of DNA by charge through the agarose sieve. Don’t wait too long Well big (long) DNA small (short) DNA dye semi-log separation distance vs MW

11. Horizontal agarose gel electrophoresis DNA For visualization staining with chemicals that bind DNA (Ethidium bromide, SYBR green, Gel red TOXIC) * * * * * * Post-stain by soaking the gel, put the dye in the gel before the run put the dye in the sample buffer

12. http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Nucleic-Acid-Detection-and-Genomics-Technology/Nucleic-Acid-Stains.html The three classes of classic nucleic acid stains (Properties of classic nucleic acid stains—Table 8.4) include:Properties of classic nucleic acid stains—Table 8.4 Intercalating dyes, such as ethidium bromide and propidium iodide Minor-groove binders, such as DAPI and the Hoechst dyes Other nucleic acid stains, including acridine orange, 7-AAD, LDS 751 and hydroxystilbamidine Figure 8.1.1 Schematic diagram showing the different binding modes of dyes (and other ligands) to DNA. RNA?

13. Horizontal agarose gel electrophoresis UV-activated fluorescence !Careful with UV! DNA Excitation 200-300nm, emission 650nm

14. TAE versus TBE, a comparison Both TAE and TBE are the common buffers used for agarose gel electrophoresis to resolve DNA fragments. Each buffer has some advantages and disadvantages, which are given below. Buffer capability : TAE has a lower buffer capacity than TBE and it tends to become exhausted during successive electrophoresis. In contrast, TBE has a more stable and higher buffering capacity. Migration of DNA :Linear, double stranded DNA migrates faster in TAE (approximately 10% faster in compare to TBE). Resolution of DNA fragments :TAE is suitable for separation of long DNA fragments while TBE is suitable for separation of small DNA fragments. Tris-borate buffer (TBE) is advantageous for obtaining a higher resolution of smaller DNA fragments on agarose gels, when compared to the conventional tris-acetate buffer (TAE). Borate buffer supports agarose cross-linking better than acetate buffer. Better cross-linking improves the sieving properties of agarose. This results in enhanced resolution of smaller DNA fragments. Recovery of DNA from agarose gel : Borate forms complex with agarose and nucleic acid. This results in low recovery of DNA from agarose gel. So if one needs to elute the DNA from the agarose gel, TAE will be better choice. Compatibility with enzyme reaction :Borate is known to be a strong inhibitor for many enzymes. So DNA eluted from Agarose-TBE gel may not be suitable for further enzymatic reaction. Integrity of DNA : The integrity of DNA sample is better in TBE buffer. This is due to presence of borate ions which can inhibits many enzymes http://bioinfoweb.com/SNDB-TAE-vs-TBE.htm

15. BUT remain careful!! Info on our webpage http://www.bioti um.com/product/ product_info/ne wproduct/gelred _gelgreen.asp

16. Interpretation 1) Molecular weight marker, shows fragment size (bp) see website, staining intensity may provide reference for amount of experimental DNA. 2) Good genomic DNA, methods used yield fragments of 20-50 kbp, RNA may be visible as a smear low in the lane 3) A smear indicates degraded DNA NO signal does NOT mean no DNA! 1 2 3

18. FLUORESCENT STAINING Ethidiumbromide drop method (Quantitative binding DNA-EthBr) known amounts DNA compare intensity of sample “dynamic range” is limited Fluorometry “Hoechst dyes” reference sample of known amount sensitive but expensive ng 1000 500 250 100 50 10 600 ng (Black box)

19. Spectrometry Chemicals in solution absorb light Depending on the chemical, Some wavelengths are absorbed more than others The amount of light absorbed depends on the concentration, transmission is reduced with increasing concentration

21. The Beer-Lambert Law: A = εlc where A is absorbance, ε is the extinction coefficient (units: M -1 cm -1 ), l is the path length that the light has travelled (units: cm) and c is concentration (units: M). http://en.wikipedia.org/wiki/Beer%E2%80%93Lambert_law Transmittance and Absorbance I T = ---- = 10 –  l = 10 –  lc I 0  = absorbance coefficient (molar absorbance x concentration) I A = - log 10 T = - log 10 (----) = - log 10 (10 –  lc ) I 0 A =  lc

22. Half the concentration, half the absorbance Compoundmax absorbance Nucleic acids 260 nm Proteins 280 nm Organic solvents230 nm A 260nm x 50 x dilution = ng/  l double stranded DNA A 260nm x 40 x dilution = ng/  l single stranded RNA 260/280 ratio of 1.8-2.0 indicates good DNA sample (low protein) watch the A 230nm for organic contamination light (x nm) diluant DNA Spectrophotometry of DNA

23. UV spectrophotometric measurement of DNA concentration and purity DNA itself, and most of the common contaminants found in DNA preps, have absorbances in the region 230nm to 320nm so measurement of the absorbances in this region allows measurement of the DNA concentration and provides information about the contaminant levels. The most important wavelengths to note are: 230nm: Guanidium salts (used to facilitate DNA binding to silica columns) and phenol (used in phenol/chloroform extractions) absorb strongly at 230nm, therefore high absorbances at this wavelength can be indicative of carry-over of either of these compounds into the sample. 260nm: DNA absorbs light most strongly at 260nm so the absorbance value at this wavelength (called A 260 ) can be used to estimate the DNA concentration using the equation Concentration (µg/ml) = (A 260 reading) × 50, which is derived from Beer’s Law.Beer’s Law 280nm: Since tyrosine and tryptophan residues absorb strongly at this wavelength, the absorbance at 280nm is used as an indicator of protein contamination. 320nm: A 320 provides a general measurement of the turbidity of the sample and is normally subtracted from the A 260 value as a background reading for the calculation of DNA concentration, but excessive values may indicate non-specific contamination. A good quality DNA sample should have a A 260 /A 280 ratio of 1.7–2.0 and an A 260 /A 230 ratio of greater than 1.5, but since the sensitivity of different techniques to these contaminants varies, these values should only be taken as a guide to the purity of your sample. http://bitesizebio.com/2007/08/22/dna-concentration-purity/

24. NANODROP: great for SMALL VOLUMES

25. SO, Do you have good/useful DNA samples? Gel results: Spec results: Never throw away samples until you tried PCR