Travails of A Scientist:Extracting DNA from Herbarium Specimens

The classification of plants and animals is based on the system devised by Carolus Linnaeus in the 18th Century. For the plant kingdom the reproductive method and flower structures are used to decide on classification. DNA represents an individual's blueprint . Recent DNA analysis has confirmed the reliability of the 18th century system.

Definition of a hybrid:

It is uncertain whether parental DNA bares any close relation to a hybrid's DNA. The following experiment was carried out on herbarium samples which had been confirmed by an expert botanist to be species plants and their hybrids. It was to test the theory that DNA analysis can confirm the identity of a plant hybrid.

Short hand used in laboratory book to describe experimentLong Hand version of laboratory bookWhat is actually happening
DNA extraction from Herbarium Specimens Deoxyribonucleic Acid extraction from Herbarium specimens Extracting the blueprint of a living individual from dried plants kept for historic reference purposes.
07/04/1998 7th April 1998 Date when experiment started, for reference purposes
RAPD analysis of Salix species and hybrids Random Amplification of Polymorphic Deoxyribonucleic Acid analysis of Salix Species and hybrids Random enlargement of the differences between DNA samples of different types of Willow tree and their crosses
Samples taken from Corstorphine Collection Small samples taken from plants in the collection made by plant collectors Robert & Margaret Corstorphine between 1884 and 1944 Samples taken from the University Museum Collections Angus Herbarium made by Robert & Margaret Corstorphine using the following plant material.
1)Salix cinera. Balgavies Loch 1912 1) Grey Willow. Collected from Balgavies Loch in 1912. 1) Grey Willow. Collected from Balgavies Loch, in 1912.
2)Salix phylicifolia. S.Esk 1937 (duplicate) 2)Tea-leaved Willow. Collected from the banks of South Esk River in 1937. Duplicate used 2) Tea-leaved Willow. Collected from the banks of South Esk River, in 1937. Duplicate used where available so catalogued specimens remain intact
3)Salix phylicifolia x cinerea S.Esk 1937 (duplicate) 3)Tea-leaved Willow crossed with Grey Willow. Collected from the banks of South Esk River, in 1937. Duplicate specimen . 3) Tea-leaved Willow crossed with Grey Willow. Collected from the banks of South Esk River, in 1937. Duplicate as above identified as hybrid by expert
4)Salix phylicifolia x cinerea S.Esk 1937 (duplicate) 4) Tea-leaved Willow crossed with Grey Willow. Collected from the banks of South Esk River, in 1937. Duplicate specimen 4) Tea-leaved Willow crossed with Grey Willow. Collected from the banks of South Esk River, in 1937. Duplicate as above
2 specimens of a hybrid used to confirm similarities.
Extraction of DNAMethod for extracting Deoxyribonucleic AcidMethod used to extract DNA
Shut leaf sample in Eppendorf tube. Put pea sized sample of leaf into small sterile plastic text tube with lid. Leaf to be handled with gloves and tweezers. Only very small piece of leaf needed. Kept sterile so they are not contaminated by human DNA
Sand added and sample ground with pestle Added a pinch of sand to the leaf sample and grind up with a small plastic rod pestle Ground to start breaking down the structure of the material
Added 500ul extraction buffer containingl
Added 500 microlitres (1000th of a millilitre) of extraction buffer made up of l
Common table saltl
Ethylenediaminetretaacetic Acid l
Sodium Dodecyl Sulphatel
Sterile distilled waterl
Tris to preserve a constant pH for the sample.l
Salt to disrupt cell walls.l
Protects DNA from digestion by any enzymes present.l
A detergent denatures proteins and weakens cell walls. l
Distilled water for dilution.l
Prevents DNA from damage by chemical reactions, such as air.
Vortexed for 5s. Incubated at room temperature for 10 min. Shaken and mixed in tube for 5 seconds left at room temperature for 10 minutes Mixed and left to soak
Centrifuged at full speed for 1 minute Spun at high speed (2000 revs per minute) at angle of 45o for one minute. Spun round to move so that sand and plant debris to the bottom of the tube
Transferred supernatant to clean tube and added 400ul phenol Poured 400 microlitres of cell solution into a clean sterile plastic tube with lid and add equal quantity of Phenol Cells are made up of many components including proteins. It is just the tiny invisible DNA cells required. The phenol starts removing all the proteins to leave the DNA
Vortexed for 2 s. Centrifuged for 5 min. Shaken for 2 seconds and returned to the centrifuge for 5 minutes Mixed and spun to separate DNA solution and the phenol (which denatures proteins), into two different layers with a layer of denatured protein between them. The DNA remains in solution.
Removed supernatant into clean tube. Added equal vol. chloroform Carefully remove upper layer (which contains the DNA in solution) into sterile tube and add equal volume of chloroform Added equal quantity of chloroform to continue cleaning the DNA solution.
Vortexed for 2s and centrifuged 5 mins. Shook for 2 seconds and returned to centrifuge for 5 minutes. Separated & extracted DNA from waste chloroform etc. Cleans off remaining Phenol and last of cell debris
Removed DNA sol. into Eppendorf tube and added equal vol. isopropanol Carefully remove upper layer of DNA in solution into sterile tube and add equal volume of isopropanol. This precipitates the DNA. Top layer of DNA solution removed.
Centrifuge 2 min. Mixed and left at room temperature for 2 min. Mixed for 2 seconds and left to soak for 2 minutes on the bench
Poured off supernatant. Poured the suspended debris off to leave the precipitated DNA This leaves the DNA on the bottom of the tube.
Resuspended DNA in 200ul h2O Diluted DNA precipitate with 200 microlitres sterile distilled water to produce DNA solution DNA on bottom of tube dissolves in water.
Added 2.5 x vol. of ethanol and vortex 2 s. Centrifuge 1 min Added 2.5 times the volume of ethanol and shaken for 2 second. Returned to centrifuge for 1 minute Added ethanol a type of 100% alcohol for a final clean of the DNA
Pour off supernatant and dry pellet under vacuum Remove ethanol off top of result and dried out to remove ethanol Dry sample in air. The ethanol evaporates.
Resuspend 50ul h2O Diluted with 50 microlitres of sterile distilled water. Put in fridge for storage till use Dilute with water to get DNA in manageable solution. Store in fridge to prevent deterioration of sample.
PCR methodPolyumerase Chain Reaction methodUsing the bundle of DNA formed, the next process makes hundreds of thousands of copies of short sections of DNA so there will be enough of each DNA fragment for it to be detected.
Set up PCR reactions with 5ul buffer l
1u dNTPs
1ul Taq
38ul h2Ol
1ul primerl
1ul DNA samplel
Total 50ul
Using prepared DNA set up a reaction for each of the 4 Salix samples
5 microlitres supplied bufferl
1 microlitres DNA basesl
1 microlitre Polymerase Enzymel
38 microlitres sterile distilled waterl
1 microlitre of a Ten base Oligonucleotide Primerl
1 microlitre DNA samplel
Total 50 microlitre for each sample
Chemicals added to DNA solution to start production of further copies of DNA strands.
PCR program
92oC 2min
35oC 2 min 
72oC 2 min
72oC 5 min
Using a 0.5 ml sterile plastic tube put the solution in a manufactured Programmable heat block and set up with the following settings
92 degrees centigrade for 2 minutes
35 degrees centigrade for 2 minutes
72 degrees centigrade for 2 minutes
Repeated 40 times
72 degrees centigrade for 5 minutes
To unzip the DNA, leaving a single strand.
Allow the ten base primers to stick to the DNA.
Allow the Polymerase to extend the primers, making short pieces of new DNA
Repeated 40 times to build up the number of each short piece of new DNA
Gel run Gel made in gel tray using a mould to create pockets to put preparation in De-ionised & purified agar jelly gel sheet with pockets formed with mould shaped like a comb.
  Preparation loaded into Gel pockets with sucrose buffer. Buffer contains a dye so the position of the invisible DNA can be seen, it is heavy so the DNA remains in the pockets and does not float off in the electrolyte fluid
  Gel on tray loaded into electrolysis machine and an electric field applied through boric acid solution An electric charge is run across the gel.
  Checked power pack is working by seeing bubbles rising and checked DNA preparation moving in correct direction. The DNA preparation moved towards the positive electrode lengthening the DNA strands. Left for 2 hours Checking that a charge is present and in the right direction Left for 2 hours
Gel stained with Ethidium Bromide.10 min. Took out Gel on tray. Gel swept into container of the dye Ethidium Bromide. Left to stain for 10 minutes - excess stained rinsed off. Gel is stained with orange dye which colours the DNA
Viewed gel Looked at gel through UV resistant goggles The stain used can only be seen with UV light. UV resistant goggles are used so the UV does not damage the eyes.
When viewed the gel failed to show any bands of DNA. Initial use of method resulted in a failure with PCR to produce bands on the gel. Nothing showed up on the prepared gel
Ran gel to see if DNA present or degraded Ran another gel with just the DNA preparation and not the PCR process to see if DNA was present and not too degraded as the specimens were old and to see how much was present Repeated the process without adding Polymerase to check if the sample of DNA produced contained enough DNA to show up on gel. Time may have degraded DNA in the Herbarium Plants so there was enough to be shown by this process.
Gel showed one very strong band from the hybrid Salix p x c, all others weak Gel showed one very strong band from the Tea-leaved Willow crossed with Grey Willow. All other bands on the gel were faint but DNA was present. Gel showed one very strong band from the Tea-leaved Willow and Grey Willow hybrid sample. All other bands on the gel were very faint showing their was DNA present, but not in large quantities.
Set up new PCR reactions with 5ul NH buffer1 yl MgCl1ul dNTPs0.1ul Taq41.9ul h2O1ul primer2ul DNA sampleTotal 50ul Set up a new reaction with slightly different chemicals5 microlitres ammonia buffer1 microlitre magnesium1 microlitres DNA bases0.1 microlitre Polymerase Enzyme41.9microlitres sterile distilled water1 microlitre of a Ten base Oligonucleotide Primer2 microlitre DNA sampleTotal 50 microlitre for each sample Tried again to create DNA strands in a gel. Used fresh Polymerase enzymes and more DNA in hope of getting better results
No result Once again on producing the gel and dying it no DNA could be seen on the gel Even though the most obvious factors that could have gone wrong were changed still no results were forthcoming. Other factors needed to be considered before the experiment was tried again and a few trials to check the chemicals being used in the process run. Some of the chemicals used are very delicate and vulnerable to being used at too high a temperature.


Part of the Made to Measure exhibition

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