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Experiment*5:*Arabidopsis!DNA$Extraction$!
During!this!lab!period!the!Arabidopsis!seedlings!will!be!observed!using!fluorescence!
microscopy.!Using!blue!to!ultra;violet!light,!GFP!will!fluoresce!green!if!it!is!expressed!in!a!
seedling.!After!observing!the!seedlings,!they!will!be!ground!up!and!DNA!extracted!to!
genotype!the!seedlings!using!PCR.!
Background++
Fluorescence+Microscopy+
The!compound!microscopes!used!in!the!
previous!lab!had!a!light!source!below!the!
specimen!that!illuminate!the!whole!
specimen.!In!contrast,!fluorescence!
microscopy!uses!an!intense!light!source!to!
excite!a!fluorescent!dye!or!protein!such!as!
GFP!from!above.!For!example!if!blue!light!
(Figure!1)!is!used!to!illuminate!a!specimen!
that!has!GFP!expression,!the!GFP!will!absorb!
the!blue!photons!and!emit!green!ones.!The!
green!light!is!then!transmitted!through!the!
objective!and!to!the!viewer.!The!remainder!
of!the!field!of!view!is!usually!dark.!The!light!
source!emits!all!colors!of!light.!Glass!filters!
are!used!to!select!the!desired!wavelength!
(excitation!filter).!The!dichroic!mirror!and!
the!emission!filter!are!used!to!block!the!
excitation!light!and!allow!the!emitted!light!
Figure!1.!Schematic!for!fluorescence!
to!pass!to!the!oculars.!This!lab!uses!a!
microscopy!optics.!Source:!
dissection!scope!instead!of!a!compound!
http://en.wikipedia.org/wiki/Fluorescence_microscope!
microscope!because!resolution!at!the!
cellular!level!is!not!needed.!The!dissection!
scope!used!in!this!lab!will!also!excite!chlorophyll!in!the!leaves!which!will!fluoresce!red.!
The!emission!filter!will!allow!the!red!light!to!pass!so!the!GFP!will!appear!as!green!
patches!on!a!red!background.!
Activity+
First!the!plants!will!be!observed!for!phenotype.!There!are!six!plants!and!each!will!be!
looked!at!using!the!fluorescence!scope.!Then!the!plants!will!be!ground!for!DNA!will!be!
extraction.!The!introduced!genes!will!be!amplified!using!primers!to!the!three!introduced!
Arabidopsis!DNA!Extraction!
!
E5.2!
genes!to!determine!the!genotype!of!each!plant.!What!genes!are!required!to!excise!
mPing!can!be!determined!using!the!phenotype!and!genotype!information.!
Viewing+GFP+Expression+
Two!weeks!ago!seeds!for!4!genotypes!of!Arabidopsis!were!planted.!They!are:!
1. Wild!Type!
2. GFP!
3. mPING!Reporter!(GFP!with!mPING)!
4. mPING!Reporter!+!Transposase!
!
An!instructor!will!demonstrate!how!to!use!the!scope.!Record!observations!in!the!lab!
notebook.!Images!will!be!made!available!on!the!course!website.!
Genotyping+the+plants+
The!seedlings!must!be!genotyped!to!determine!which!genes!are!present!in!the!GFP!
expressing!plants.!This!is!done!by!first!extracting!the!genomic!DNA!from!each!line.!
Second!PCR!with!primers!against!the!transgenes!(GFP!and!Transposase)!will!be!done!to!
determine!what!genes!are!present.!
DNA+Extraction+
The!DNA!extraction!will!be!started!today,!but!possibly!not!finished.!A!stopping!point!will!
be!announced!in!class.!
!
Each!group!of!two!will!extract!DNA!from!the!4!lines!(each!partner!does!2!extractions).!
!
/Materials/list:/
Extraction!Buffer!!
10%!SDS!(sodium!dodecyl!sulfate)!
5M!KOAc!(Potassium!Acetate)!
3!pieces!of!Miracloth!(filter!cloth)!
100%!Isopropanol!
70%!Ethanol!
Resuspension!Buffer!
Ice!
65˚C!bead!block!
1.5!ml!Tubes!
2!ml!screw!cap!tubes!
Metal!beads!
!
Method:/
1. Label!1!tube!with!the!plant!name!and!your!initials.!!
!
2. Harvest!2;3!seedlings!and!place!in!a!screw;capped!tube.!!Add!1000!µl!of!
Extraction!Buffer!and!3!metal!beads.!The/Extraction/buffer/contains/EDTA/a/
chemical/that/removes/magnesium/ions/from/solution/and/prevents/DNases/from/
Arabidopsis!DNA!Extraction!
!
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/
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E5.3!
destroying/the/DNA./The/buffer/keeps/the/pH/slightly/basic/to/further/block/
enzyme/function.!
3. Place!on!bead!shaker.!Shake!for!10!minutes.!
4. Add!120!µl!of!10%!SDS.!Mix!by!inverting.!SDS/is/a/detergent/that/denatures/and/
binds/to/the/hydrophobic/portion/of/protein/and/lipids./SDS/is/the/main/detergent/
in/shampoo/where/it/does/the/same/thing/to/clean/hair./
Prepare/all/samples/to/this/step./Keep/them/on/ice/until/all/are/ready/for/step/5./
!
5. Place!tubes!in!a!65˚C!bead!block!for!20!minutes.!
!
6. Add!300!µl!5M!KOAc.!!Mix!well!by!inverting!several!times!(important!),!then!
place!on!ice!5!minutes.!KOAc/causes/the/SDS/to/precipitate/along/with/the/
proteins/and/lipids./The/solution/will/turn/cloudy./!
7. Spin!for!5!minutes!at!top!speed!in!a!centrifuge.!Label!a!new!tube.!
8. Squirt!about!800!µl!of!the!supernatant!through!miracloth!funnel!into!the!labeled!
1.5!ml!tube.!The!instructors!will!demonstrate!how!to!make!a!funnel!out!of!the!
miracloth./This/step/removes/solids/that/did/not/pellet/during/the/centrifugation./
9. Add!600!µl!of!isopropanol.!Mix!the!contents!thoroughly!by!inverting.!!The/
isopropanol/(rubbing/alcohol)/causes/a/change/in/the/hydrophobicity/of/the/
solution/and/the/DNA/precipitates.!DNA/precipitate/may/or/may/not/be/visible/at/
this/point;/don’t/worry/if/you/don't/see/much./Potential/stopping/point.!
10. Spin!for!5!minutes!at!top!speed.!/Look/for/a/small/glassy/pellet/in/the/bottom/of/
the/tube/that/may/be/visible./The/instructors/will/help/you./!
/
11. Carefully!pour!off!supernatant!Then!use!a!P20!set!to!20!µl!to!remove!the!last!
drops.!
!
12. Add!500!µl!of!70%!ethanol!and!flick!until!the!pellet!comes!off!the!bottom.!The/
ethanol/removes/residual/isopropanol/and/salts/from/the/pellet./Potential/
stopping/point.!
13. Spin!5!minutes.!
14. Pour!off!the!ethanol.!Then!use!a!P20!set!to!20!µl!to!remove!the!last!drops.!!Make!
sure!the!pellet!stays!in!the!tube!!!
Arabidopsis!DNA!Extraction!
!
!
E5.4!
15. Place!the!tube!in!the!bead!bath!with!the!top!open!for!5!minutes!to!allow!the!
remaining!ethanol!to!evaporate.!
16. Resuspend!the!DNA!in!50!µl!Resuspension!Buffer.!Let!sit!at!RT!for!about!5!
minutes.!Depending!on!amount!of!starting!material!may!need!to!be!diluted!for!
PCR.!
Learning+Goals+
!Students!will!be!able!to:!
1. Compare!and!contrast!bright!field!microscopy!with!fluorescence!microscopy.!
2. Interpret!the!images!from!the!microscopy!in!relation!to!TE!activity.!
3. Extract!DNA!from!plants.!
!