PCR is DNA replication in a test tube Ex. 25: PCR Based Testing for Water Contaminants Day 2

Materials needed per table:  Amplified PCR products from last session  Bottle of 0.5 X TBE (Tris- borate/EDTA) buffer  50 ml measuring cylinder, 250ml Erlenmeyer flask  Minigel electrophoresis apparatus with gel tray, spacers, and comb  micropipettes and matching tips  Microcentrifuge tube containing 20 µl of 10x gel loading buffer. Materials shared by whole class: 1.Scales, flasks, electrophoresis grade agarose, cylinder, and TBE buffer at “gel making station” 2.Microwave 3.Power source (4 available per class) 4.Nanofuge (2 per room) 5.DNA standard (instructor handles it) 6.“InstaStain” ethidium bromide staining sheets (handled or supervised by the instructor). 7.Gel documentation system Day 2 Cast, Load, and Electrophorese a 1.5 % Agarose Gel

Understanding Gel Electrophoresis Make 30 ml of a 1.5% agarose gel. How?

How does gel electrophoresis separate DNA fragments? Gel acts as sieve to filter DNA fragments DNA fragments are naturally negatively charged (phosphate backbone) DNA pulled towards anode (pos. electrode) by electric attraction Smaller fragments move faster through the gel and larger fragments move slower gel electrophoresis is optimized by adjusting agarose concentration in gel

DNA is negatively charged and therefore repelled from the negative pole and attracted towards the positive pole

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A Typical Image of an Agarose Gel Under UV Light Decreasing DNA Size Largest DNA fragments Smallest DNA fragments

The Intensity of the Band is Proportional to the Concentration Of DNA An important point to remember is that the intensity of the band is proportional to the amount of DNA found in the band The upper band has far less DNA when compared to the lower band. The intensity of the bands are proportional to the amount of DNA at that position in the gel

Sizing The Fragments of DNA The sizes of the various fragments can be identified by including a “ladder” in the gel –A ladder is a mixture of DNA fragments of known size –A ladder is usually run beside the unknown sample so that the sizes of the various DNA fragments in the sample can be identified

Marker / Ladder / Size Standard Mixture of DNA fragments of selected sizes When run in a gel, fragments will separate into distinct bands that can be used as references Fragment size always stated as [X] base pairs (bp) Two common ladders: 200bp and 1kbp (1000 bp) ladders 200 bp ladder composed of a mixture of small fragments (200 to 4000 bp) Ladders commercially available

Sizing a Gel Product Base Pairs (bp) Kbp Sample ladder 2000 bp 1000 bp Sample 1Kbp ladder Size of this Fragment?

Size In Base Pair (bp) The size of the fragment is…?? ? 1 kbp ladder was used

Music videoMusic video from "Scientists for Better PCR" and Bio-Rad.