Protocols

• Flow chart for sequencing bryophyte DNA
• Isolation of DNA from Fresh or Dried Moss Tissue
• DNA extraction from agar cultures
• Agarose gells
• Setting Up PCR Reactions
• Cleaning amplicons for sequencing using shrimp alkaline phosphatase (SAP)
• Sequencing PRC - Big Dyes
• Preparing GenBank submissions with Sequin
• TOPO-TA cloning
• AFLP Protocol

Basic Lab Procedure for Sequencing Bryophyte DNA

Isolate DNA

Run small aliquot on agarose gel to check quality and quantity of DNA, or use Nanodrop

Amplify DNA with appropriate primers (PCR)

Run 2-5µl PCR product on agarose gel to check for amplification of desired product

Clean PCR product with shrimp alkaline phosphatase (SAP)

Perform sequencing PCR

Ready for automated sequencer

Isolation of DNA from Fresh or Dried Moss Tissue

A. Grinding and extracting with CTAB Buffer

1. Turn on water bath to 60°C.
2. Prepare fresh CTAB isolation buffer by adding 0.2% b-mercaptoethanol (BME, brown bottle in hood) to 2X stock CTAB buffer (warm under tap water to get in solution). For 25 samples: 17.5µl CTAB & 35µl BME, work in hood.
3. Use round bottom tubes & one steel ball/ea for Genodrinder.
4. Use Genogrinder - Liquid Nitrogen Protocol to grind plant material. Open tubes quickly/carefully after removing from liquid nitrogen to let off pressure in tubes. Grind samples 1'30'', 100 strokes/min. Return to lab with samples.
5. Pipette 700µl of CTAB buffer without delay. Work out of hood since it will suck up your ground samples.
6. Vortex all samples.
7. Put all tubes in water bath at 60°C for 30-60 min. Invert tubes periodically during incubation.

Meanwhile: label TWO new sets of eppendorf 2µl tubes.

B. Removal of proteins with chloroform:IAA

1. Add an equal volume (700µl) of chloroform:IAA (24:1) to plant extract. If more needed, mix up 96ml Chl and 4ml of IAA. Add in hood.
2. Cap tubes securely and mix gently by inversion to produce an emulsion. You can mix this just by putting another test tube rack on top. Invert 50 times, more or less, to produce emulsion.
   Be carefull with the Chl:IAA because this can take off the labels, and it leaks out of pipetter.
3. Centrifuge 1-5 minutes at 10,000 rpm in room temperature.
4. Remove the aqueous (top) phase to a clean labeled tube. AVOID THE INTERFACE!! Dump the Chl:IAA and the bead into the funneled waste and tubes and pipette tips into the hood waste. About 500µl will be the amount you pipette off this stage.
5. Grade all tubes into roughly equivalent amounts (300-400-500-600).

C. Precipitation, washing and drying of DNA (out of hood from here on)

1. Add an equal volume of cold isopropanol (300-400-500-600) to aqueous phase. Mix gently by inversion (5-6 times). Precipitate at -20°C for 20+ min. Can precipitate overnight if necessary.
2. Centrifuge for 10 min. at 13,000 rpm. Pour or aspirate off supernatant. Your DNA is in the pellet!!
3. Take pellets to SpeedVac and leave drying there for 5 minutes at room temperature (10minutes seems to be too much). You can use incubator for 2-3 hours if you want.
4. Resuspend the pellet in 20-50µl dH2O or TE.
5. Incubate for 5 minutes in the heat block at 60°C until pellet dissolves (the cleaner the product is, the easier it resuspends).

D. (GeneClean Protocol)

1. Turn on temp block to 50°C.
2. Add 3 volumes of NaI (i.e. to 30µl of DNA, add 90µl NaI).
3. Be sure that glassmilk tube is well vortexed before using. Use a different tip for each addition, since the tips get clogged.
4. Add 5µl glassmilk to DNA/NaI solution. Put the tip right down into the NaI so the glassmilk goes into the solution.
5. Flick tube gently with your finger, and incubate at room temperature (RT) for 5-15 minutes (just set in tray). Flick tube gently every 1-2 minutes during this incubation.
6. Spin down in microfuge (full speed, 5 seconds) to pellet glassmilk.
7. Remove and discard NaI supernatant (pipette) well inside the waste container. Eject the tip inside the the waste container as the iodine stains everything.
8. To glassmilk pellet add 700µl cold New Wash. Let sit at RT for 5 min. while it washes the DNA.
9. Remove New Wash by just pouring it off carefully. The pellet still has the DNA.
10. Repeat New Wash rinse (step 8-9) two more times (for a total of three changes of New Wash).
11. After removing New Wash the third time, spin for 5 seconds at >8000 rpm and pipette off any remaining traces of New Wash.
12. Resuspend washed glassmilk pellet in 20µl TE buffer per tube.
13. Incubate the tubes with buffer in 50°C water bath or temp block for about 2 minutes.
14. Centrifuge the tubes for 30 seconds and move supernatant (about 20µl, now the pellet is glass and supernatant contains your DNA) to a clean labeled tube. Avoid interface!
15. Store at -20°C until ready to run an agarose gel to determine concentrations (optional).
16. TURN OFF TEMP BLOCK!

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DNA Extraction from Agar Cultures

Preparation of samples:

* Cut thin strips of upper layer of media or chunk of media with fungal growth and place into eppendorf tube. Having more mycelium and less agar is ideal. Try not to use more than approximately an 1/8" cube.
* Tightly cap tubes and keep samples on bench until use. Avoid waiting more than 2-3 days to avoid drying of the agar.

Phase one:

1. Add 500 µl SDS Extraction Buffer to each eppie.
2. Let samples sit at room temperature for 3-4 hours.
3. Grind samples to a soup consistency with sterile micropestles.
4. Let samples sit overnight at room temperature.

Phase two:

1. Add 500 µl phenol:chloroform:IAA to each sample tube (kept in freezer, let warm to room temp and make sure it is homogenous).
2. Tightly cap tubes and invert several times to produce an emulsion. Centrifuge at full speed (13,000 rpm) for 15 minutes. While the samples are centrifuging, label clean eppies.
3. Remove the supernatant to clean tubes, being careful to avoid the interface. 300-400 ul is realistic.
4. Further clean the samples by adding 250 µl of chloroform:IAA to each tube. Tightly cap tubes and invert several times to produce an emulsion. Centrifuge at full speed (13,000 rpm) for 15 minutes. While the samples are centrifuging, label clean eppies.
5. Remove the supernatant to clean tubes, being careful to avoid the interface. 200-300 µl is realistic.
6. Add 0.6 vol. of cold 100% isopropyl alcohol (from freezer) to each tube to precipitate DNA.
   (200 ul -> 120 µl; 300 µl -> 180 µl; 400 µl -> 240 µl; 500 µl -> 300 µl).
7. Invert tubes several times, then put into freezer for 5-10 minutes.
8. Centrifuge tubes for 15 minutes at full speed.
9. Carefully pour off the supernatant (the pellet at the bottom of the tube may or may not be visible, but it is the DNA).
10. Add 500 µl of cold 70% ethanol (from freezer).
11. Centrifuge for 5 minutes at full speed.
12. Carefull pour off the ethanol.
13. Dry tubes completely in a Speed-vac (~30 min. at room temperature)
14. Resuspend DNA in 40 µl TE.

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Agarose Gels in the Shaw Lab

1) Pouring a gel:

• Prepare the gel tray by placing it in the buffer chamber with the gasketted ends firmly sealed against the black walls of the chamber. If the chamber is clean and empty, push the tray to the bottom of the chamber, between the two plastic bumps. If there is usable buffer in the chamber, position the tray 1-2cm below the upper edges of the chamber (above the buffer). In both cases, ensure that the tray is level using the bubble thing. If you're using the midi- or maxi-gel, you can use the casting rig.
• Place the desired combs in the slots of the gel tray.
• Measure desired quantity of agarose on top-loading balance. Use the Amresco Agarose I from the shelf above the balance.
• Transfer agarose to EtBr-designated Erlenmeyer flask (250ml flask for mini-gel quantity, 500ml flask for midi- or maxi-gel quantity).
• Measure the appropriate volume of 1X buffer in the TAE-designated graduated cylinder.
• Pour approximately HALF of this volume into the flask containing the powdered agarose.
• Plug the flask opening loosely with a bunched-up Kimwipe.
• Heat the flask in the microwave for approximately 1min, stopping at around the 30sec point to swirl the flask well (beware of escaping steam!).
• After 1min, swirl the flask again, holding it up to a window or light to ensure that there are no bits of undissolved agarose in the liquid. If there are still visible "lenses" of material, continue heating and swirling in 10sec increments til the solution is uniform.
• Place the flask on the EtBr-bench stir plate. Set the plate to stir very slowly (setting 1-2).
• Add the remainder of the buffer volume to the stirring flask.
• Add the appropriate volume of EtBr to the stirring liquid, using the dropper bottle from the shelf above the EtBr bench. EtBr is NASTY (mutagenic, etc.) and light sensitive. Don't handle anything on the EtBr bench without gloves! Return the EtBr bottle to the dark container when finished.
• If the bottom of the flask still feels hot to your gloved hand (i.e.>60°C), allow it to cool another 5-10 minutes on the stir plate. Very hot agarose can warp the gel tray!
• Gently pour the liquid into the prepared gel tray, taking care to avoid bubbles, and preventing the stir bar from plopping down on the tray surface.
• If major bubbles are visible, these can be removed with a pipet tip or a Pasteur pipet. The combs can be removed for this purpose as long as they are replaced before polymerization begins.
• Allow the gel to polymerize at least 30min. Carefully remove tray from buffer chamber (or casting rig), turn 90° and replace (submerging in buffer, if it is already present in the chamber). If reusing buffer, rock the chamber back and forth a couple of times to mix. Differences in pH can develop in the buffer at the positive and negative ends of the chamber after extended electrophoresis. The buffer should be replaced every 3-4 uses.
• Supporting the edges of the acrylic tray (hold down with one hand), apply a gentle upward motion to the comb. Taking care not to tear the wells, remove both combs.
• Ensure that buffer completely covers the gel (no "dimples" over the wells), but do not fill more than necessary. Too much buffer over the gel can retard electrophoresis.

2) Loading a gel:

• Cut a 3-5in piece of parafilm. Place it on the bench paper film-side up, and place two black rubber gel dams along the edges to hold the film down.
• Using a p20 pipetman, draw up nµl of blue loading dye, where n is the number of samples you have. If you have more than 10 samples, do this is multiple aliquots.
• Place n blue "dots" in a line (or multiple lines) on the parafilm, making each approximately 1µl in volume. The concentration of the loading dye is 6X, so that for every 5µl of aqueous sample to be loaded, 1µl of loading dye gives the appropriate density and color to the aliquot.
• In an order that will be easily remembered by you, place 5µl of each sample to be loaded in a separate blue dot, pipetting up and down to mix if you like. Use a new tip for each sample!
• When all of your dots are mixed, begin transferring them to the wells of the submerged gel, again in an order you will know later. Write it down NOW if you need to! It can be useful to place one of the black rubber gel dams under the chamber so that the wells are visible against the dark background.
• With the sample in the tip of the pipetman, hold the tip so that the end is just inside the desired well. Slowly push the plunger of the pipetman in to dispense the blue sample. It should sink into the well.
• Rinse the pipet tip in the clean buffer at the end of the chamber and proceed with the next sample. It is not necessary to change tips between samples at this point (but never go into your stock or PCR tube with a dirty tip!)
• When all samples are loaded, add 1µl of the size marker (fX/HaeIII fragments) to an adjacent empty lane.
• Place the cover on the chamber, taking care not to bump the gel tray. Ensure that the red/positive lead is at the end toward which you intend your samples to run. Plug the leads into the appropriate connections on the power supply. Turn on the power and adjust the voltage to a maximum of 8V/cm (the distance is that between the electrodes, not the length of the gel itself). For the mini-gel, you can run the gel at 105V or less. This is a good running voltage for the midi-gel also, but you can go as high as 140V if you're really in a hurry. If the side of the gel chamber gets warm (>30°C), turn down the voltage. High temperatures can distort or melt the gel!
• Ensure that the gel is running by checking for the presence of rising bubbles near the wires of the electrodes. Also double check that the leads are orientated so that your DNA is running towards the positive. The DNA will run off the end of the gel if the leads are reversed!
• Run the gel until the bromophenol blue of the loading dye has migrated the desired distance. Bromophenol blue migrates at about the same rate as a 300bp fragment of double-stranded DNA. For most of our purposes, 2-3cm migration distance on a mini-gel is sufficient for good band separation (takes "20min at 95V). There's plenty of room for greater separation on a two-tiered midi-gel. The size marker will look better and be more useful if you're patient.
• Turn off the current at the power supply and remove the top of the gel chamber. Remove the tray containing the gel to an EtBr-designated container for transport to the UV-transilluminator.

3) Visualizing DNA bands:

• For optimum visualization, place the gel directly on the surface of the transilluminator. The gel tray is UV transparent, and you can leave the gel on the tray if you desire (works very well for maxi-gel trays!).
• Turn on the three units of the Eagle-Eye camera system. Turn on the UV source, and without looking at the transilluminator (bright UV will harm your eyes!), close the doors to the box.
• Make the necessary adjustments to optimize your view of the gel on the monitor. Adjust the size and focus of the gel image with the rings on the camera lens. Adjust the brightness of the image with the "integration" counter on the frame integrator/storage unit, and the "brightness" and "contrast" buttons on the copy processor unit. When you're happy with the appearance of the gel, move the lever on the copy processor unit to "print", and push the "print" button to get the printout of the gel image.
• Return the gel carefully to the tray if it is going to be reused, or dispose of it in the EtBr gel waste container near the Eagle-Eye.

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Setting Up PCR Reactions

* provided by Taq manufacturer
** 0.5mM each: dATP, dTTP, dGTP, dCTP
† when stock DNA is of concentration appropriate for use as indicated

Doing It:

A. To minimize pipetting and maximize accuracy, make a Master Mix containing all PCR components except template DNA:

i) Multiply the quantities for the desired reaction size by the number of reactions you are setting up. Include a positive and negative control, and at least one reaction volume extra, to account for pipetting error.

ii) Add reaction components to a clean Eppendorf tube in the order indicated above.

iii) Vortex the master mix well and flick with finger, holding tube against light to make sure solution is well mixed. Spin down in mini-centrifuge to collect contents in bottom of tube.

iv) Aliquot appropriate quantity of master mix (14µl or 19µl) to clean, labeled PCR tubes or a PCR plate. You can do this with one pipet tip since the reaction tubes are clean. You can also use the electronic repeater pipet, if you are setting up lots of reactions.

B. Add the appropriate quantity of template DNA, placing the pipet tip at the bottom of the tube and dispensing the DNA into the master mix.

C. Cap the tubes or seal the plate with film, and spin briefly to collect all of the contents in the bottoms of the tubes/wells.

D. Initiate thermocycling with the appropriate program.

For regular PCR amplification:

Screen Says or Select:	You Press:
Run	Enter
A: Sandy	Enter
A: 03 STD50	Enter
Hotlid Auto	Enter
Loading Alarm Off
End Run Alarm Off	Enter
Lid on during final hold step? No	Enter
Tube	Right arrow
Sim Tube	Enter
Number of samples	# tubes you have, Enter
Sample Volume	your sample volume, Enter
Lid preheat before auto start	Nothing - you're done!

E. Place tubes in block and close thermocycler lid.

Notes:

• Remove reaction components from freezer awhile before you plan to set up your reactions. Vortex to mix before use and hold on ice. Taq polymerase does not freeze solid, so should be kept on ice the entire time.
• It can be important that reaction components be kept cold prior to the initiation of thermocycling. Nonspecific amplification products can be produced at intermediate (≈RT) warm temperatures. Keep PCR reaction tubes in a cold rack until they are placed in preheated thermocycler.
• The quantity of DNA being added to these reactions is VERY arbitrary. It is only necessary that you get an amount that will be sufficient to amplify, yet not so much that the reaction is overwhelmed with inhibitors. Don't sweat the 1.0-1.3 µl measurement! You just want to get a smidgen in there. If your PCR is unsuccessful, DNA template quantity is something to be manipulated, usually by diluting the DNA and trying a lesser quantity in the reaction.
• The other reaction component that can be manipulated to increase amplification efficiency is the MgCl2 concentration. You can optimize this by setting up a series of reactions with MgCl2 concentrations ranging between 1.0mM and 4.0mM. Adjust the amount of water in the reaction to compensate for the greater or lesser amount of MgCl2.
• For the negative control, just place an aliquot of master mix in a reaction tube and add water in lieu of DNA. Do this last, when most opportunities for contamination have passed and can be detected. The positive control is not required but can be VERY helpful. For a positive, use an aliquot of template DNA that has been previously shown to amplify for the gene of interest (borrow one, if necessary!).
  

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Cleaning amplicons for sequencing using Antarctic Phosphatase (AP)

1. For each 17 ul PCR (from a 20 ul reaction, with 3 ul removed for checking on a gel), add:
   
   • 0.3 µl Exonuclease I
   • 0.3 µl Antarctic Phosphatase
   • 2.7 µl sterile water to bring volume up to ease pipetting
2. Place in thermalcycler and run "clean" (machine 1) or "shrimp" (machine 2) under lab folder.
   This program runs for a total of 30 minutes:
   
   • 15 minutes @ 37°C
   • 15 minutes @ 80°C

Et voila, products are now ready for use in sequencing PCR!

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Sequencing PCR - Big Dyes

1. Thaw the desired primers (10µM concentration) and the Big Dye Buffer, and place on ice. Briefly thaw the "sequencing juice" (PE Applied Biosystems Big Dye Terminator Rxn. Mix). Gently mix and place on ice.
2. Place a PCR-tube rack in ice or use a "cold rack" from the freezer. Label PCR tubes sequentially and place in rack in a grid formation. Orient your primers in the ice at the top of the rack, and cleaned DNA's in the ice along the left side of the rack (SEE GRID DIAGRAM NEXT PAGE).
3. For each primer, label a tube for "master mix". Into it place, per reaction (or n+1, to have a little extra):
   
   • 1µl of Big Dye sequencing juice
   • 2µl of Big Dye Buffer
   • 1.0µl of sterile ddH2O
   • 1.0µl of primer
4. Aliquot 5.0µl of master mix into the tubes in the appropriate column (refer to grid diagram). Place the mix in a dot on the sidewall of the tube. You can aliquot each column (all the same primer) with a single clean pipet tip.
5. Aliquot 5.0 µl each DNA* to the tubes in the appropriate row, avoiding tip contact with your master mix dot. Use a separate tip for each DNA (obviously!).
6. Turn on the Hybaid PCR Express and initiate the appropriate cycle**.

For sequencing PCR amplification:

Screen Says or Select:	You Press:
Run	Enter
A: Sandy	Down arrow, 4 times
E: Lab	Enter
E: 04 Seq	Enter
Hotlid Auto	Enter
Loading Alarm Off
End Run Alarm Off	Enter
Lid on during final hold step? No	Enter
Tube	Right arrow
Sim Tube	Enter
Number of samples	# tubes you have, Enter
Sample Volume	your sample volume, Enter
Lid preheat before auto start	Nothing - you're done!

7. Place tubes in block and close thermocycler lid.

* If your quantified cleaned DNA is more concentrated than 5-15ng per 5µl (for PCR product), use less DNA and make up the volume with sterile water. Water can be added as a third component (master mix+water+DNA to total of 10µl), or dilutions of DNA can be made so that 5µl dispensed to the sequencing reaction contains an appropriate amount of DNA.

**on a Hybaid PCR Express:
25 cycles: 10 sec/96°C ; 5 sec/50°C ; 4 min/60°C.

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Preparing GenBank Submissions with Sequin

S.B.B. 10/6/99, Updated 8/15/02

The NCBI shareware program Sequin, is designed for the preparation of GenBank sequence submissions off-line. It is a stand-alone program that users can download for use on their personal computers. As a final step in the preparation of a submission, Sequin writes a file that the user then emails directly to the GenBank folks as an attachment. Sequin walks you through the preparation of the submission, for the most part, but it has a tendency to crash. There are also many points at which the next "step" is not intuitive or clearly prompted. These instructions are meant to alleviate some of these difficulties for users wishing to submit an aligned matrix of sequences as a "batch", thus eliminating the need to prepare each sequence singly. The great advantage of this method is that Sequin can "propagate" features (such as the beginning and end of a gene or intron), over your aligned matrix, placing the boundaries for each individual sequence at the correct base.

1. Prepare your Nexus file:

Prepare a Nexus file according to the format of the attached sample. Be certain that each taxon has a corresponding "definition" line appearing in the correct order, in brackets and started with a ">", at the end of the matrix. A code name for the taxon/sample, that will serve as a temporary accession number until one is assigned, should appear directly above its corresponding line of sequence, flush left. The code name must begin with a letter (not a number!!). Be certain that the number of taxa (NTAX) and the number of characters (NCHAR) in your sequence alignment as stated at the top of your Nexus file are correct. All coding (such as 1s and 0s to indicate presence/absence of indels and ? marks) must be removed from the matrix, taking care to maintain the alignment. If Ns have been added to the ends of some sequences for the purposes of maintaining an alignment, these must remain. The Genbank guy assures me that they remove these later, so even lots (100's!) of Ns appended to the end(s) are O.K. As a final step, view your Nexus file in Word with all formatting shown. Remove any extraneous formatting (e.g. spaces at the ends of code names, not visible when viewed in Paup!) that may mess up reading of the file by Sequin. Save your Nexus file as something that will be easily recognized as formatted for Sequin.

2. Launch Sequin and begin preparing submission:

In the Shaw Lab, Sequin can be launched by selecting the Sequin alias under the Apple/Applications pull-down menu. Choose the "Start New Submission" for a GenBank submission on the initial window. Enter submission information (manuscript title, release date if not immediate, contact, authors and affiliation). One can move back and forth among these "pages" with the buttons at the bottom, but the next "form" cannot be reached until all of these fields have been completed.

3. Load sequence matrix into Sequin:

On the Sequence Format window of the next form, choose "Phylogenetic Study" and "Contiguous", if you have prepared your matrix like the sample (this is FASTA+GAP/NEXUS). On the Organism and Sequences form, you can choose the location of the sequence (i.e., genomic, chloroplast, etc.), from the pull-down menu. Choose "Standard" for the Genetic Code for Translation (also pull-down). Proceed next to the Annotation page (skip Nucleotide for now!). Here, one can add a title (such as "nuclear ribosomal DNA internal transcribed spacer", etc.), to be appended to each of your definition lines (now only an organism name as specified in your original matrix). Select "Prefix title with organism name" to make this happen. Return to the Nucleotide page (one previous) and click "Import Nucleotide Contiguous". Choose your Nexus file in the usual Mac/Menu method, and pray if you are so inclined. It is only after you've loaded your matrix that Sequin will allow you to save ANY of the work you've done so far. If it crashes while loading, you're screwed and you have to start over. It pays to have your Nexus file done right the first time (see above). If your matrix loads successfully, you'll get a window indicating how many sequences, etc. Thank your lucky stars and proceed to the next form, which is Modifiers. I suggest pulling down "lineage" under the "Modifier" Menu. You can then enter, for each of your sequences, the lineage information in the usual format (e.g. Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Bryopsida; Sphagnidae; Sphagnales; Sphagnaceae; Sphagnum). Include the semicolons between each taxonomic designation, but no period is necessary at the end. The program puts the period in for you. Cutting and pasting between the cells on this "spreadsheet" can expedite this process. The lineage information will apear after the species name in the "Organism" field of you Genbank entry. The organism name itself comes from the definition line you should have put in your Nexus file (see above). Click O.K.

Now you should get a window with your first sequence shown in GenBank format. Save now or you will regret it forever!!! Don't ever copy over this file, if you don't want to weep bitter tears later. Call it "ITSprelim.sqn" or something and keep it for posterity. Changes you make from here on will be largely undoable, as far as I can tell, so if you mess up, or if the thing crashes, you'll want to abandon the bad one and revert to this pristine saved version. Starting over from here is much better than starting over from the beginning.

4. Basics of sequence display formats:

At this point, you are likely looking at a window containing the GenBank format of your first sequence. You can change the Display Format with the pull-down menu. The most useful options other than the "GenBank" view are the "Sequence" and "Alignment" views. You can select individual sequences to view in the "Target Sequence" scroll-down menu, or you can select "All Sequences", and see them in a consecutive list. The GenBank view is a good place to check that the sizes for your different sequences are correct. The sizes will be in base pairs after the program has removed the gaps from the sequence as it appeared in the Nexus matrix. The Sequence view gives you a nice numbered display of the nucleotide sequence, and this is the best place to start adding features (i.e. delineating genes, introns, exons, etc.) to your sequences. Beware! In the sequence view as it initially appears, the program seems to have a bug, that makes it impossible to scroll down in the window. Strange pink lines appear when you try to do this. The problem is eliminated by clicking on the [enlarge window/shrink window] button in the lined bar at the top of the window (looks like a square within a square). It seems necessary to be at the "top" of the sequence when you enlarge the window or only the lower portion of the sequence will be visible (another bug). This done, the window will fill the screen, and your sequence in its entirety should look fine. Tinker back and forth with this if things look funny. Seeing a buggy view doesn't seem to cause permanent harm to the file.

5. Annotating features:

It's important to note that features are defined in a single sequence (the first one in your matrix), in the Sequence view mode just described. This will be your "Reference Sequence", in which features are assigned according to the base numbers in the sequence as you see it in its "degapped" form (i.e. not gapped for alignment). Features are then propagated to the other sequences in the aligned ("gapped") matrix in a different view (see below). After propagation, Sequin calculates the different positions for the features for each individual sequence in the alignment.

To annotate features, in the enlarged Sequence view for your first sequence (reference sequence), pull down the "Annotate" menu, then "Genes and Named Regions...". The other seemingly useful option, "Coding Regions and Transcripts..." has some problems that I haven't figured out, so I've been sticking to calling things a gene or a region (which you can then give a name). To illustrate the process, say you're dealing with a matrix of its sequences. You want to annotate ITS1, 5.8S, ITS2, and the 5' end of the 26S. Note the numerical positions for these regions in your reference sequence. It's helpful to write down the series of 6-8 bases that begin and end each region, so you can check the Sequin-assigned positions for the other sequences later. For ITS1, pull down the "Annotate" menu, then "Genes and Named Regions...", then "Region". Type "ITS1" for name, leave the Properties page as-is, and give the range of base numbers on the Location page. Then click "Accept". A black arrow delineating the region should appear on your Sequence view. Proceed with the other regions/genes. For genes (such as 5.8S), I've been putting the name in the Locus field. Otherwise the entry is completed in the same manner. For a region where the sequence is not complete (such as for the 26S, where the 3' end may be incomplete), click "3' (or 5') partial". When Sequin assigns base numbers to this region, they will appear with a < or >. When you're done annotating features for your reference sequence, you're ready to propagate (the fun part!).

6. Propagating features:

In the Target Sequence scroll-window, highlight "All Sequences". Then change the Display Format to Alignment. In the initial Alignment view, you'll see a bunch of weird stuff, with a red box around it. Position the cursor in the middle of the red box and double click. Be prepared to wait patiently as Sequin prepares the view. The clock icon may or may not indicate that the computer is "thinking". Sometimes the arrow icon is visible even when it's still completing a command. Patience is a virtue. You should now see your aligned sequence matrix, with gaps and all, with your reference sequence at the top. To see the features you've assigned to the reference sequence applied to it's aligned form (with gaps), click the "Show feat." button at the bottom of the window. Check to see that the regions are still correctly annotated (by looking at the nucleotide sequence -- base numbers will be different!!) for the reference sequence. Dashes in the black line should indicate where the delineated feature is "skipping over" gaps. The feature in view should be named under the taxon code name at the left. The moment of truth...under the Features menu, choose "Propagate". Select your single Source (reference) sequence, for which you've already defined the features. In the other scroll-window, click the "Select all" button, then holding down the shift key, deselect your reference sequence. Hit the "Select all" button for the Source features (or select the subset you want to propagate). Be sure the "extend over gaps" button is selected and hit the "Propagate" button. Again, be patient, and you may need to scroll up or down before changes (features now shown for all sequences in the alignment) will be visible. It's not too useful to continue viewing all sequences in this way, so you can "Dismiss" (button at the bottom) this window and switch to Sequence or GenBank display format for a single sequence. Here you can check to see that the propagated features have been placed correctly. You are now ready to "Prepare Submission..." under the file menu. This is what you send, as an email attachment, to gb-sub@ncbi.nlm.nih.gov. Within a day or two, you should receive an email reply containing GenBank accession numbers assigned to your temporary taxon codes.

A final note...while it is difficult to successfully change much of the information you entered in the early part of this process, there are some useful tidbits under the Edit menu, that can be accessed throughout. "Edit Submitter Info..." allows you to add additional contact information, or to give special instructions to the database staff. Good Luck!

Additions or amendments to these instructions are welcome!!!

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TOPO-TA Cloning the Vilgalys-lab Way

Preparation:

• Equilibrate water bath to 42°C.
• Warm vial of SOC to room temperature.
• Warm LBamp, x-gal plates (1 per transformation) at 37°C.
• Turn on shaking incubator (37°C).

One-fifth "Cloning Reaction":

• To a labeled 0.5 ml tube, add:
  0.8 µl fresh PCR product or concentrated, cleaned PCR product
  0.2 µl salt solution
  0.2 µl vector
  (final volume: 1.2 µl)
• Spin briefly, if necessary, and mix gently.
• Incubate 5-30 min at room temperature (maybe 20min optimal?).

Transformation:

1/3 transformation reaction, X = 17µl cells
1/4 transformation reaction, X = 12.5µl cells
1/5 transformation reaction, X = 10µl cells

• Thaw ON ICE one tube of TOP10 competent cells per set of 3-5 transformations.
• With a wide-bore pipet tip, transfer X µl from the tube of cells to each of two labeled, round-bottom Eppendorf tubes, leaving approximately X µl in the original tube.
• Add 1 µl of each ligation/cloning reaction to a separate, labeled tube of cells, stirring very gently.
  !!Do not mix by pipetting up and down!!
• Incubate on ice for 5-30 min. (no advantage to longer times).
• Heat shock cells for 30 sec at 42°C without shaking.
• Immediately transfer tubes to ice. Let sit 2 min.
• Add 150 µl of room temperature SOC medium to cells.
• Cap tubes tightly and shake HORIZONTALLY (200 rpm) at 37°C for 1 hour.
• Spread all of the cells on a single labeled LBamp, x-gal plate. Incubate overnight at 37°C.
• Screen white colonies for presence of the desired insert by touching a colony with a toothpick and "inoculating" a prepared PCR reaction.
• Pick screened colonies to a fresh LBamp, x-gal plate, and grow overnight at 37°C. Parafilm plate edge and refrigerate for long-term storage.

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AFLP Protocol

Materials

Adapters:

EcoRI site:
5' CTCGTAGACTGCGTACC
      CATCTGACGCATGGTTAA 5'

MseI site:
5' GACGATGAGTCCTGAG
       TACTCAGGACTCAT 5'

To make double-stranded adapters from oligonucleotides:

• Purchase oligos as described above, with bases read from 5' to 3'.
• Mix equal moles of the two complementary oligos in a tube (i.e. mix equal volumes of equal concentrations -- higher concentrations work better!).
• Incubate in boiling water bath for 2 min.
• Allow tube to cool to room temperature. Annealing occurs as the tube cools.

PCR Primers:

Primer	Core sequence	Enzyme-specific sequence	Selective base(s) (1 to 3)
EcoRI-X	5' GACTGCGTACC	AATTC	XXX 3'
MseI-X	5' GATGAGTCCTGAG	TAA	XXX 3'

Available:

Purchased by Shian-Ren	Purchased by Sandy
EcoRI-GT unlabeled	EcoRI-G
EcoRI-GT w/ FAM label (blue)	EcoRI-C
EcoRI-CA unlabeled	EcoRI-A
EcoRI-CA w/TET label (green)	MseI-A
EcoRI-CAG unlabeled	MseI-T
EcoRI-AG w/HEX label (yellow)	MseI-G
MseI-AC
MseI-TG
MseI-TGC
MseI-GA
MseI-GT

10X AFLP Buffer:

Tris-HAc pH 7.5	100mM
Mg(Ac)2	100mM
Kac	500mM
DTT	50mM
BSA	0.5mg/ml

Note: a large quantity of this buffer was made by Shian-Ren. It is stored frozen and is available for use.

 

Procedure

I. AFLP RE Digests - make cocktail for multiple ligation reactions:

volume per reaction	full size	1/4 size
ddH20	q.s. to 40µl	none
10X AFLP Buffer	4µl	1.0µl
EcoRI	0.7µl	0.2µl
MseI	1.3µl	0.3µl
DNA	200-500ng	8.5µl
Total Digest Volume	40µl	10µl

• Use full size recipe when DNA quantity is not limited. Use 1/4 size recipe for weak/limited DNA samples.
• Incubate reactions at 37°C for 3h.

II. AFLP ligation - make cocktail for multiple ligation reactions:

volume per reaction	full size	1/4 size
ddH20	5.75µl	1.44µl
10X AFLP Buffer	1µl	0.25µl
10mM ATP	1µl	0.25µl
T4 DNA ligase	0.25µl	0.06µl
5µM d.s. EcoRI adapters	1µl	0.25µl
50µM d.s. MseI adapters	1µl	0.25µl
Total Ligation Volume	10µl	2.5µl

• Add full ligation volume (10µl or 2.5µl) to correspondingly-sized digest (40µl or 10µl).
• Incubate reactions at room temperature for 3h.
• Make 1:5 dilution of each ligation reaction.

III. Preamplification (Optional) - make cocktail for multiple reactions:

volume per 25µl reaction
ddH20	14.65µl
10X PCR Buffer w/o MgCl2	2.5µl
2mM dNTPs	2.5µl
50mM MgCl2	0.75µl
"EcoRI" primer	1.0µl
"MseI" primer	1.0µl
Taq Polymerase	0.1µl

• Add 22.5µl cocktail to 2.5µl diluted ligation reaction.
• Amplify according to the following thermocycler specifications:

File Type	Denaturation Time/Temp.	Annealing Time/Temp.	Extension Time/Temp.	#Cycles
Step Cycle	30sec/94°C	60sec/56°C	60sec/72°C	20

IV. AFLP amplification:

volume per 25µl reaction
ddH20	13.65µl
10X PCR Buffer w/o MgCl2	2.5µl
2mM dNTPs	2.5µl
50mM MgCl2	0.75µl
"EcoRI" primer	2.5.µl
"MseI" primer	2.5.µl
Taq Polymerase	0.1µl

• Add 24.5µl cocktail to 0.5µl undiluted preamplification reaction.
• Amplify according to the following thermocycler specifications:

File #	File Type	Denaturation Time/Temp.	Annealing Time/Temp.	Extension Time/Temp.	#Cycles	Linked to File#
1	Step Cycle	1min/94°C	1min/65°C	90sec/72°C	1	2
2	Step Cycle	1min/94°C	1min/64°C	90sec/72°C	1	3
3	Step Cycle	1min/94°C	1min/63°C	90sec/72°C	1	4
4	Step Cycle	1min/94°C	1min/62°C	90sec/72°C	1	5
5	Step Cycle	1min/94°C	1min/61°C	90sec/72°C	1	6
6	Step Cycle	1min/94°C	1min/60°C	90sec/72°C	1	7
7	Step Cycle	1min/94°C	1min/59°C	90sec/72°C	1	8
8	Step Cycle	1min/94°C	1min/58°C	90sec/72°C	1	9
9	Step Cycle	1min/94°C	1min/57°C	90sec/72°C	1	10
10	Step Cycle	1min/94°C	1min/56°C	90sec/72°C	1	11
11	Step Cycle	30sec/94°C	30sec/56°C	1min/72°C	23	4°C soak

V. Visualizing AFLP products:

• Run 3-5µl of AFLP amplification product on a high percentage agarose gel or acrylamide gel, to check for signs of successful amplification and/or variation.
• If AFLP amplification was performed with a fluorescently-labeled "EcoRI" primer, products may be visualized on an acrylamide gel run on the ABI 377 automated sequencer.

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 Shaw Laboratory
 139 Biological Sciences Bldg.
 Box 90338
 Department of Biology
 Duke University
 Durham
 North Carolina 27708
 U.S.A.

 Phone: (+1) 919 660-7345
 Fax: (+1) 919 660-7293