National Centre for Biotechnology Education The PCR and Plant evolution Copyright © Dean Madden,...
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National Centre for Biotechnology Education www.ncbe.reading.ac.uk The PCR and Plant evolution Copyright © Dean Madden, 2012
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Transcript of National Centre for Biotechnology Education The PCR and Plant evolution Copyright © Dean Madden,...
National Centre for Biotechnology Education
www.ncbe.reading.ac.uk
The PCR and
Plant evolution
Copyright © Dean Madden, 2012
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Stroma
Outer membrane Inner
membrane
Starchgranule
Granum
Stromalamellae
Lipidglobule
DNA within the chloroplast
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Passed on in ovules
No recombination
Highly conserved(only insertions, deletions, and substitutions)
120–150 kb
Encodes ~80 proteins
Essential for photosynthesis
Chloroplast DNA
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RuBisCo
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AngiospermPhylogeny Group
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RuBisCo — DNA sequence data
AGP
DNA encoding tRNA — Stable, in all plants
Non-coding regions — High mutation rate
NCBE/SAPS kit
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tRNAtRNA tRNAtRNANON-CODINGNON-CODING
300–500 bp
Variation in the size of this region
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Active site here strips primers from the DNA
DNA is made at this active site
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Taq polymerase
Non-target DNA
Target DNA
Primers
50–65 °CPrimers anneal to complementarysequences of bases in the single-
stranded target DNA
72 °CTaq DNA polymeraseMakes double-stranded DNA, using the single strands as templates
94–98 °CThe double-stranded DNA
is split into two strands
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Taq polymerase
Non-target DNA
Target DNA
Primers
Start First cycle Second cycle Third cycle Fourth cycle
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Mass A microgram is one millionth of a gram
1 000 micrograms (µg) = 1 milligram (mg) 1 000 milligrams = 1 gram (g)
Volume
A microlitre is one millionth of a litre 1 000 microlitres (µL) = 1 millilitre (mL)
1 000 millilitres = 1 litre (L)
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Soft rubber tubing
Yellow graduated tip
HOLD HEREDo not touch the point!
10 µL
20 µL
50 µL
100 µL
Measure to the top of each
band
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Microsyringe
Graduated tip
HOLD HEREDo not touch the point!
10 µL
2 µL
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Fixed volume micropipetteUse yellow tips to dispense 20 µL volumes
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Summary of the procedure
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Place the leaf tissue on the card
Ensure that it fits within the box
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Close the cover and squash the
leaf
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Write the plant’s name in pencil on
the cover
Allow the card to dry for one hour
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Cut discs using a punch
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Push the disc from the punch using some plastic ‘wire’
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100 µL
Wash the disc twice in purification reagent
Add 100 µL of purification reagent
Flick to mix
Remove the liquid
REPEAT
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100 µL
Wash the disc twice in TE-1 buffer
Add 100 µL of TE-1 buffer
Flick to mix
Remove the liquid
REPEAT
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Primer 110 µL
Primer 210 µL
Water4 or 6 µL
PCR ‘bead’, containing:
– Taq polymerase – buffer – dNTPs – magnesium chloride
Primer 1: 5’–CGAAATCGGTAGACGCTACG–3’Primer 2: 5’–GGGGATAGAGGGACTTGAAC–3’
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Use forceps to add a disc to the plant DNA
Label the tube
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30 seconds
30 seconds
30 seconds
Repeat this three-step cycle
30 times
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Frosted panel on this side
Molten agarose55–60 °C
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Cut two electrodes
Carbon fibretissue
42 mm
22 mm
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Pour 2–3 mm depth of buffer over the gel before you ease the comb out
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Mix loading dye into each
DNA sample
2 µL
Bromophenol blue loading dye
AmplifiedDNA sample
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Label the end of the tank to show the contents of
each well
Black card under the tank reveals
the wells for loading
Load the DNA through the buffer, taking care not to puncture the wells as you do so
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Electrodes
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Direction of DNA movement
Place a comb over the tank to reduce
evaporation
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Leave the stain on for 4 minutes only
Azure A stain
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DNA
Positively-charged Azure A binds to the negatively-charged
phosphate groups of the DNA
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Area with DNA bands
Wells Loading dye
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DNA ‘ruler’ or ‘ladder’
Sizes are in kb
