November 2001
Sheffield Molecular Genetics Facility
Semi-automated fluorescent genotyping
by
Andrew Krupa ()
Nic Chaline ()
Disclaimer
We have provided this protocol as a courtesy, in the hope that this will help you in your research. Although we have taken great care to record it accurately, we do not accept any responsibility for the accuracy or for any costs or damages that you might suffer as a consequence of mistakes in this protocol. If you use this protocol it is implicit that you accept the conditions of this disclaimer.
Please acknowledge the Sheffield Molecular Genetics Facility when using this protocol.
Contents
Preparation of samples from blood to PCR product
Timetable
I)Safety- hazards and signing of COSHH formsDay 1. p.m.
II)DNA extraction (Ammonium acetate precipitation method, Bruford et al. 1998)Day 1. p.m.
III)Assessing the quality and quantity of DNA (using a 0.8% agarose gel and fluorometer)Day 2. a.m.
IV)Setting up a PCR reaction (including reaction recipes etc.)Day 3. a.m.
“GeneScanning” your samples
I)Cleaning the plates and putting the gel ‘cassette’ togetherDay 4. a.m.
II)Preparing the gel mixDay 4. a.m.
III)Pouring the gelDay 4. a.m.
IV)Setting up a sample sheetDay 4. a.m.
V)Final preparation of samplesDay 4. p.m.
VI)Running the gel (plate check, pre-run and run)Day 4. p.m.
VII)Loading the samplesDay 4. p.m.
VIII)What to do after the gel has runDay 5. a.m.
Data preparation- using GeneScan 2.1
I)Using GeneScan software to prepare your samples for genotypingDay 5. a.m.
Data preparation- using GenoTyper
I)Using GenoTyper software to create your final GenotypesDay 5. p.m.
(I)
Safety- hazards and signing of COSHH forms
There are numerous hazards that we are going to encounter in the lab. For these everyone needs to read and sign relevant COSHH forms. Above all show common sense and GLP (good laboratory practice). Ensure that you wear appropriate safety ware (lab coats (fastened up!), gloves and safety glasses).
Specific hazards
Acrylamide is a neurotoxin and carcinogen
Ethidium Bromide is a carcinogen and teratogen
Proteinase K
(II)
DNA extraction from blood/tissue (Ammonium acetate precipitation method, Bruford et al. (1998))
1)Place in a 1.5ml flip-top tube 250ul Digsol buffer and 10ul Proteinase K (10mg/ml). Keep on ice.
2)Centrifuge blood sample at 13,000rpm for about 1 min.
3)Remove sample from ethanol with toothpick and blot onto clean tissue. When dry, transfer the toothpick into the tube and jiggle to dislodge the blood. Remove toothpick and place in bleach
4)Vortex, wrap the rack in tissue and cling-film and place in rotating oven at 55 (3hrs) or 37 degrees centigrade (O/N).
5)Once digested (straw colour) add 300ul 4M ammonium acetate to each sample
6)Vortex several times over a period of at least 15 minutes at room temp.
7)Centrifuge for 10 minutes at 15,000rpm
8)Aspirate supernatant (clear liquid) into clean labelled 1.5ml flip-top tubes (discard the gunky protein stuff)
9)Add 1ml 100% ethanol
10)Invert tubes gently several times to precipitate DNA
11)Centrifuge for 10 minutes at 15,000rpm
12)Pour off ethanol taking care not to lose DNA pellet
13)Add 500ul 70% ethanol and invert several times to rinse pellet
14)If the pellet dislodges from the bottom of the tube centrifuge for 5 minutes at 15,000rpm
15)Pour off ethanol and stand tubes upside-down on clean tissue (approx. 30 minutes)
16)Once fully dry add 100ul T10 E0.1. (the amount added is dependent on size of pellet)
17)Flick sample to dislodge pellet
18)Place tubes in waterbath for 30 minutes (37 or 65 degrees) to dissolve pellet (flicking every 10 mins)
19)Store at –20 degrees (long term) or 4 degrees (short term)
Other DNA extraction methods are commonly used using phenol/chloroform, Chelex resin and prepared kits. Protocols are available for these methods and it will be possible if there is time to try some of these methods.
Bruford MW, Hanotte O, Brookfield JFY, Burke T (1998) Multilocus and single-locus DNA fingerprinting. In: Molecular Genetic Analysis of Populations: A Practical Approach, 2nd edition, (ed. Hoelzel AR), pp. 287-336. IRL Press, Oxford.
(III)
Assessing the quality and quantity of DNA (using a 0.8% agarose gel and fluorometer)
Making an agarose gel
1)Place masking tape at the ends of a 20cmx20cm plastic gel tray. Place four 27-well combs in position. Place on a flat surface
2) Make up a 0.8% agarose gel (250ml 1xTBE, 1.75 g agarose) in a 500ml flask. Heat in microwave (NB. take care as it becomes superheated. Stir every minute to release the bubbles). Add 9ul ethidium bromide (1mg/ml) and cool under a tap whilst rotating the flask
3)Pour into the prepared plastic gel tray
4)Leave to set for approx. 30 minutes
5)When set remove combs and place in electrophoresis tank containing 1XTBE buffer
Preparing samples
1)Pipette 14ul 1x Orange G loading buffer into wells into a 72-well Terrasaki plate
2)Add 1ul of the sample
3)To the last well add 0.5ug of Lambda DNA standard
4)Load all the sample on the gel
5)Electrophorese the gel (100Volts, DNA is –ve so goes toward the +ve electrode).
Quantification of DNA using a DNA fluorometer (Hoefer DynaQuant200)
1) Switch on the fluorometer and allow it to warm up for 20 minutes
2) Add 2ml of fluorometer dye solution to a clean cuvette. Place in the fluorometer
2)Press “Zero”
3)Remove the cuvette and add 2ul of 100ng/ul calf thymus DNA
4)Place in the fluorometer, press CALIB, type in 100 then press ENTER. The value should read “100”
5)Remove the cuvette, rinse with 2ml of fluorometer dye solution
6)Add 2ml of fluorometer dye solution to the clean cuvette. Place in the fluorometer
7)Press “zero” if necessary. Add 2ul of your sample.
8)Read off the value given as ng/ul. This is the DNA concentration of your sample
9)Repeat from step 5 with all your samples
10)You can now dilute all your samples to “PCR concentration” i.e. 10ng/ul by removing 10ul of sample to a new tube and adding as water the concentration in ng/ul –10. This will make a 10ng/ul stock
i.e Fluorometer value of 210. Remove 10ul and place in a tube, add 200ul of water. Mix. This is makes a 10ng/ul working solution.
(IV)
Setting up a PCR reaction
1)Clean your area, pipettes etc with 10% bleach (can use UV if available)
2)Add the reagents to a 1.5ml tube. The values in the boxes- next page!-are calculated for 1 sample. Multiply by the no. of PCR reactions you want to do + 10% to allow for pipetting inaccuracies. The calculations for most of the different MgCl2 concentrations are given on the next page for reference.
3)If you are testing a lot of primers at the same MgCl2 concentration then make up a big “mastermix” for all samples without the primer. Then split this into “sub-mastermixes” before adding the primer.
4)Once the mastermix is prepared you can aliquot your DNA into wells in a microtitre plate
5)Add 9ul of the mastermix to each DNA sample
6)Add a drop of oil, cover in Saranwrap and place in the PCR machine on the correct temperature profile
Fridolfsson AK and Ellegren H (1999) A simple and universal method for molecular sexing of non-ratite birds. Journal of Avian Biology, 30, 116-121.
Griffiths R, Double MC, Orr K and Dawson RJG (1998) A DNA test to sex most birds. Molecular Ecology, 7, 1071-1075.
(IV) Cont.
PCR reaction recipes- all volumes in ul
(V)
Checking PCR reactions on 2% agarose gel (resolves most 2550F/2718R products)
Making an agarose gel
6)Place masking tape at the ends of a 20cmx20cm plastic gel tray. Place four 27-well combs in position. Place on a flat surface
7) Make up a 2% agarose gel (250ml 1xTBE, 5 g agarose) in a 500ml flask. Heat in microwave (NB. take care as it becomes superheated). Add 9ul ethidium bromide (1mg/ml) and cool under a tap whilst rotating the flask
8)Pour into the prepared plastic gel tray
9)Leave to set for approx. 30 minutes
10)When set remove combs and place in electrophoresis tank containing 1XTBE buffer
Preparing samples
6)Pipette 10ul of 2x Orange G loading buffer directly into the samples (it will drop under the oil).
7)To an empty Terrasaki plate add 0.5ug of 123bp ladder as a standard (14ul 1xOrange G/ 0.5ul 123bp)
8)Load all the samples on the gel
9)Run at 100 volts (DNA is -ve charged and will migrate toward the +ve electrode). After 1 hour we can look at the gel and visualise the DNA under UV light on a transilluminator and produce an image for analysis
Analysis of the results
1) With 2550F/2718R one band is produced in male birds and two bands in females
NB. For routine checking of PCR products pre-GeneScanning we would test a number on a 1.5% agarose gel (take 5ul product out and add to orange G and run on a 1.5% gel). With most primer systems which are ‘behaving’ you wouldn’t need to do this.
“GeneScanning” your samples using an ABI 377 DNA Sequencer
(I)
Cleaning the plates and putting the gel cassette together
•Clean all the equipment glass plates, cassettes, combs and spacers. Always use hot tap water (resting plates on foam). Rinse well with top quality distilled water (necessary with plates only).
•Dry plates by standing at 60 degrees gel-side down on white tissue (so dust doesn't fall on it). Pay particular attention to the gel side-indicated by a vertical etched line or a 'V' on the top of the plate pointing to the gel-side. Don't dry gel-side with tissue (unless very careful not to get dust on it). Note: the 96-well eared plates have a channel cut out at the top of the gel-side. The other equipment can be dried well with tissue.
Make the gel mix (applies to all well-to-read sizes and all comb types)
•Only use clean water. Detergents etc. may cause unwanted fluorescence.
•Place the cassette on a hard white drip tray (ensure that this is spotlessly clean).
•Place the long plate on bottom- gel-side up after checking that it is totally dry (down to metal/plastic prongs near laser read bit).
•Put spacers flush to the side and bottom (notch side-in and up towards the top - use a spot of quality water on the spacer to help it stick to the plate).
•Put on the eared plate (ears to the top-make sure if you are wanting to do 96 wells that you use the correct plate- the one with a frosted cut-out bit a the top) and ensure that the plates are flush with the bottom.
•Clamp up cassette clamps to the top.
•Place rig so that it is is on the edge of the drip tray and that it angles down toward the top of the plate.
(II)
Preparing the gel mix
You are advised to degas the bolded parts of the gel before adding the APS and TEMED (although we don't do this!!)
These communal solutions are pipetted using communal pipettes. Clean the pipettes before and after use by rinsing in top quality water. The urea is aliquoted out (see Rota) as it is a suspected mutagen. If you use the last pot, aliquot some more out in the fumehood
5% 24cm wtr
•Urea 8.4g
dd Water 12.2ml
10XTBE (filtered) 2.4ml
Long Ranger (50%)2.4ml
Dissolve the above ingredients for 10 minutes before adding the TEMED and 10% APS(wear purple gloves and goggles).
Add the following catalysts in the fumehood
10% APS 117ul
TEMED 16.5ul
•Mix all this on a stirrer (be careful not to introduce bubbles into the gel mix). Label your beaker as Toxic.
(III)
Pouring the gel
•Suck up into a reusable syringe (be careful to prevent bubbles) then slowly pour the gel (firstly, drip a few drops into the top left between the plates. Tap the glass as it goes between the plates). You should have a good 4 minutes to do it.
•Insert in the flat casting comb (for 96-well 0.4mm rather than 0.2mm used for 48 well gels- these are marked)
•Place a bulldog clip at the top (protecting the glass with a piece of tissue).
•Leave to set (1 hour minimum, 24 hours max.)
(IV)
Setting up a Sample Sheet
•Open up the hard disc.
•Open up ABI 377XL Collection folder.
•Open up ABI 377XL Collection.
•Under File choose New and select GeneScan Sample. This is the sample sheet that you will use for each of your sample files. Fill in the Sample Name with something that individually identifies each sample clearly. Do this for all 96 of your samples. Give the sheet a name and save it into the sample sheet folder (you are advised to do this in advance). If you don't do this you'll need to use the 96 well blank sheet. If you duplicate a sample sheet you can then change it and use a new name.
(V)
Final preparation of the samples
•PCR samples should be stored in the freezer (the dye may 'drop off' in time if you don't freeze them- after about 1 week??). Remove them from the freezer and if you are multiplexing (mixing all loci together into one loading) you will need to work out the dilution factor, band sizes and "messiness" for each of your loci (you will have to test a selection of the products independently on test gels to get experience of how your alleles behave).
•We aren’t doing ‘multiplexing’ today (bold parts).To multiplex ensure that the samples are diluted with water in situ (add with the Gilson Distriman pipette). Take 5ul of plate 1 first row (using a 12-channel pipette). Rinse the tips once in a tray of clean top quality water. Proceed to collect all the row 1 samples from your other loci with these same tips. Discard the tips and proceed with row 2 samples. Take 1.8ul of the mix and place it into 1.5ul of formamide/TAMRA or ROX internal standard in a 96-well plate (use a Distriman to aliquot the loading buffer).
This is usually made up as follows
• formamide85ul
ABI TAMRA/ROX35ul
dextran blue35ul
•formamide600ul
Genpak TAMRA/ROX180ul
loading buffer200ul
• formamide150ul
HomeBrand TAMRA/ROX35ul
loading buffer35ul
HomeBrand is cheaper and the amounts of standard could be dropped further if you are sure that each of your standard peak heights is at least 100-150 (ask if you are unsure). For new projects you should opt for this one.
Ensure that you are using the correct dye standard for your set. If your set includes primers labelled with NED then you must use filter set D modules and ROX standard. For TAMRA sets use filter set C.
(VI)
Running the gel (Plate Check, Prerun and Run.)
•After the gel has set (check that this has happened to the excess in your beaker/syringe) wash the gel plates to get rid of any polyacrylamide and pay particular attention to the laser-read area (if you have done a clean pouring job it won't need cleaning) and the comb area (take the comb out and clean any bits of gel out with a combination of a stream of water, tissue and an old comb). Spend a few minutes doing a good job here as this will save real hassle later on. Dry the plates.
•When you are satisfied that everything is fine take the gel to the machine (now is a good time to switch the machine and computer on- it is sometimes a good idea to restart the computer).
•Put the bottom buffer tank in place.
•Place the gel in the machine, clamp it in place (including the black 'laser bar' at the bottom). Close the door.
•Open up the hard disc.
•Open up ABI 377XL Collection folder.
•Open up ABI 377XL Collection.
•Go to the Window bar
•Go to Gel Preferences
•Go to Default File Names
•Open this up and change the Run File and the Run Folder names to the name of your gel (8 characters).
•Choose New under File and select GeneScan Run. The Run File will now open.
•Ensure that you have selected the correct run modules, no. of samples, sample sheet and run time (Ask about this).
Basically if you have got any NED labelled loci you should use filter set D (ROX standard) if not then use filter set C (TAMRA standard). Make sure that you choose
1)the correct no. of samples
2)the correct size of plate
3)the correct run and pre-run modules
4)the correct sample sheet
5)a suitable run time for the size of your plate and your allele sizes
The sheet on the right of the machine will give you this information (if you are in any doubt about this then ask).
•Select Plate Check (wait a few minutes and under Window you will be able to check on the Status of the machine- laser power, electricity- also you will be able to look at the Scan Window and the Gel Image)
If the Scan Window shows a nice and flat horizontal line(s) then you can carry on (if not it is probable that there is some rubbish on the outside of your gel and you will have to take it away from the machine and clean it. Sometimes there is a solid object, bubble or contaminant in the gel itself and depending on it's nature it may disappear before your alleles appear).
•If OK select Terminate from the Run Window
• Open the door of the machine.
•Attach the top chamber (put 0.5XTBE into the top chamber so that it floods the comb area- there is a black pen mark as a filling guide).
•Check that the top chamber is not leaking.
•(for 36 cm gels only- Clamp in the white heat-transfer plate and attach all the electrodes and water pipes (put a bit of water around the rubber 'O' rings)).
•Close the door and select Prerun in the Run Window. This will warm the gel up and run out some impurities in the gel. Have a look at the Scan, Gel Image and Status. You should aim to load your samples when the gel has reached 51 degrees (about 15 minutes). Don't leave it any longer as the gel distorts at the top (if you need more time select Pause which switches off the electricity but still keeps the gel warm).
•Heat up your prepared samples to 94oC in a Hybaid PCR machine for 4 minutes (you can do it for longer if you want to concentrate your samples more). Quench and keep on ice for the duration of loading. 'Squoosh' the wells on the gel to get rid of excess urea using a plastic pasteur pipette with yellow tip attached.