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National Centre for Biotechnology Education www.ncbe.reading.ac.uk The Lambda protocol Copyright © Dean Madden, 2012.

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Presentation on theme: "National Centre for Biotechnology Education www.ncbe.reading.ac.uk The Lambda protocol Copyright © Dean Madden, 2012."— Presentation transcript:

1 National Centre for Biotechnology Education The Lambda protocol Copyright © Dean Madden, 2012

2 E. coli Lambda phage Lambda DNA E. coli DNA Lambda DNA enters cell and forms a ring Lysogenic cycleLytic cycle Activation UV light Lambda DNA integrates within the host E. coli chromosome Lambda DNA replicated Lysis E. coli bursts open, releasing new lambda phages

3 Copyright © Dean Madden, 2012 Head with DNA Tail Lambda DNA base pairs Non-essential region HeadTail Integration of DNA into bacterial chromosome Early control DNA synthesisLate controlLysis of bacterium

4 Copyright © Dean Madden, 2012 BamHI

5 Copyright © Dean Madden, 2012 BamHI

6 Copyright © Dean Madden, 2012 EcoRI

7 Copyright © Dean Madden, 2012 HindIII

8 Copyright © Dean Madden, 2012 BamHI HindIII EcoRI

9 Copyright © Dean Madden, 2012

10 Summary of procedure

11 Copyright © Dean Madden, 2012 Microsyringe Graduated tip HOLD HERE Do not touch the point! 10 µL 2 µL

12 Copyright © Dean Madden, 2012 Mass A microgram is one millionth of a gram micrograms (µg) = 1 milligram (mg) milligrams = 1 gram (g) Volume A microlitre is one millionth of a litre microlitres (µL) = 1 millilitre (mL) millilitres = 1 litre (L)

13 Copyright © Dean Madden, 2012 Add 100 µL of distilled water 30 µg of dried lambda DNA Cap the tube firmly Flick the tube repeatedly to dissolve the DNA

14 Copyright © Dean Madden, 2012 Mix the DNA solution thoroughly before dispensing it Lambda DNA solution 20 µL Use a new tip to add the DNA solution to each tube, to prevent contamination EcoRIBamHIHindIIIEmpty

15 Copyright © Dean Madden, 2012 Close the tubes and push them firmly into the foam floater Incubate for 30–45 minutes at 37°C

16 Copyright © Dean Madden, 2012 Cut two electrodes Carbon fibre tissue 42 mm 22 mm

17 Copyright © Dean Madden, 2012 Frosted panel on this side Molten agarose 55–60 °C

18 Copyright © Dean Madden, 2012 Pour buffer over the gel before removing the comb

19 Copyright © Dean Madden, 2012 Mix loading dye into each DNA sample 2 µL

20 Copyright © Dean Madden, 2012 TBE buffer solution Label the end of the tank to show the contents of each well Black card under the tank reveals the wells for loading 2–3 mm depth of buffer over the gel

21 Copyright © Dean Madden, 2012 Electrodes

22 Copyright © Dean Madden, 2012 Direction of DNA movement Place a comb over the tank to reduce evaporation

23 Copyright © Dean Madden, 2012 Leave the stain on for 4 minutes only Azure A stain

24 Copyright © Dean Madden, 2012 DNA Positively-charged Azure A binds to the negatively-charged phosphate groups of the DNA

25 Copyright © Dean Madden, 2012 Area with DNA bands WellsLoading dye Millimetre graph paper beneath the gel is a convenient way to measure distances

26 Copyright © Dean Madden, 2012 Specimen results EcoRIBamHIHindIIIUncut


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