pcr is dna replication in a test tube ex. 25: pcr based testing for water contaminants day 2

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.