pcr is dna replication in a test tube ex. 25: pcr based testing for water contaminants day 2
TRANSCRIPT
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 2Cast, 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
_ _ _ _ _ _ _ _ _ _ _ _
+ + + + + + + + + + + + +
A Typical Image of an Agarose Gel Under UV Light
Decreasing DNASize
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 lessDNA when compared to the lowerband. 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
BasePairs(bp)4000300020001600
1000
500
1Kbp Sample
ladder
2000 bp
1000 bp
40003000
20001600
1000
500
Sample 1Kbp
ladder
Size of this Fragment?
100
200
400
300
600
1200
1500
1000
500
Size In Base Pair (bp)
The size of the fragment is…??
40003000
20001600
1000
500
?
1 kbp ladder was used
Music video from "Scientists for Better PCR" and Bio-Rad.