Basic Lab Procedure for Sequencing
aliquot on agarose gel to check quality and quantity of DNA, or use Nanodrop
DNA with appropriate primers (PCR)
Run 2-5µl PCR product
on agarose gel to check for amplification of desired product
PCR product with shrimp alkaline phosphatase (SAP)
Ready for automated sequencer
Isolation of DNA from Fresh or Dried Moss Tissue
Grinding and extracting with CTAB Buffer
- Turn on water bath
- 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.
round bottom tubes & one steel ball/ea for Genodrinder.
- 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.
- Pipette 700µl of
CTAB buffer without delay. Work out of hood since it will suck up your ground
- Vortex all samples.
- Put all tubes in water bath at 60°C
for 30-60 min. Invert tubes periodically during incubation.
label TWO new sets of eppendorf 2µl tubes.
B. Removal of proteins with
- 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
- 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.
- Centrifuge 1-5
minutes at 10,000 rpm in room temperature.
- 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.
all tubes into roughly equivalent amounts (300-400-500-600).
Precipitation, washing and drying of DNA (out of hood from here on)
- 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.
- Centrifuge for 10 min. at 13,000
rpm. Pour or aspirate off supernatant. Your DNA is in the pellet!!
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.
the pellet in 20-50µl dH2O or TE.
- 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)
- Turn on temp block to 50°C.
- Add 3 volumes of NaI (i.e. to 30µl of
DNA, add 90µl NaI).
- Be sure that glassmilk tube is well vortexed before
using. Use a different tip for each addition, since the tips get clogged.
5µl glassmilk to DNA/NaI solution. Put the tip right down into the NaI so the
glassmilk goes into the solution.
- 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.
- Spin down in microfuge
(full speed, 5 seconds) to pellet glassmilk.
- Remove and discard NaI supernatant
(pipette) well inside the waste container. Eject the tip inside the the waste
container as the iodine stains everything.
- To glassmilk pellet add 700µl
cold New Wash. Let sit at RT for 5 min. while it washes the DNA.
New Wash by just pouring it off carefully. The pellet still has the DNA.
New Wash rinse (step 8-9) two more times (for a total of three changes of New
- After removing New Wash the third time, spin for 5 seconds at
>8000 rpm and pipette off any remaining traces of New Wash.
washed glassmilk pellet in 20µl TE buffer per tube.
- Incubate the tubes
with buffer in 50°C water bath or temp block for about 2 minutes.
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!
at -20°C until ready to run an agarose gel to determine concentrations (optional).
- TURN OFF TEMP BLOCK!
DNA Extraction from Agar Cultures
* 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.
cap tubes and keep samples on bench until use. Avoid waiting more than 2-3 days
to avoid drying of the agar.
- Add 500 µl
SDS Extraction Buffer to each eppie.
- Let samples sit at room temperature
for 3-4 hours.
- Grind samples to a soup consistency with sterile micropestles.
- Let samples sit overnight at room temperature.
- Add 500 µl phenol:chloroform:IAA to each sample tube (kept
in freezer, let warm to room temp and make sure it is homogenous).
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
- Remove the supernatant to clean tubes, being careful to avoid
the interface. 300-400 ul is realistic.
- 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.
- Remove the supernatant
to clean tubes, being careful to avoid the interface. 200-300 µl is realistic.
- 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).
- Invert tubes several times, then put into freezer
for 5-10 minutes.
- Centrifuge tubes for 15 minutes at full speed.
pour off the supernatant (the pellet at the bottom of the tube may or may not
be visible, but it is the DNA).
- Add 500 µl of cold 70% ethanol (from freezer).
- Centrifuge for 5 minutes at full speed.
- Carefull pour off
- Dry tubes completely in a Speed-vac (~30 min. at room temperature)
- Resuspend DNA in 40 µl TE.
Agarose Gels in the Shaw Lab
| || |
|Vol. 1X TAE(ml)|| |
| || |
|50 (mini-gel)|| |
|80 (midi-gel)|| |
|180 (maxi-gel)|| || |
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
- Place the desired combs in the slots of the gel tray.
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
- 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
- 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!
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
- 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.
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.
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
- 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
- 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
- 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.
- 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!).
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.
Setting Up PCR Reactions
Conc. in Rxn.
Quantity (µl) in 15 µl Rxn. †
Quantity (µl) in 20µl Rxn.
|50mM MgCl 2
|primer 1 (10µM)||
|primer 2 (10µM)||
|Taq Polymerase (5units/µl)||
provided by Taq manufacturer
** 0.5mM each: dATP, dTTP, dGTP, dCTP
stock DNA is of concentration appropriate for use as indicated
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.
regular PCR amplification:
|Screen Says or Select: ||You Press:
|Run ||Enter |
|A: Sandy ||Enter|
|A: 03 STD50 ||Enter
|Hotlid Auto ||Enter|
Off || |
|End Run Alarm Off ||Enter|
|Lid on during final hold step? No ||Enter|
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.
- 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!).
Cleaning amplicons for sequencing using Antarctic
- For each 17 ul PCR (from a 20 ul reaction,
with 3 ul removed for checking on a gel), add:
- 0.3 µl Exonuclease
- 0.3 µl Antarctic Phosphatase
- 2.7 µl sterile water to bring volume up to ease pipetting
- Place in thermalcycler and run "clean" (machine 1)
or "shrimp" (machine 2) under lab folder.
This program runs for a total of
- 15 minutes @ 37°C
- 15 minutes @ 80°C
voila, products are now ready for use in sequencing PCR!
Sequencing PCR - Big Dyes
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.
- 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
- 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
- 2µl of Big Dye Buffer
- 1.0µl of sterile ddH2O
- 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
- 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
- Turn on the Hybaid PCR Express and initiate the appropriate
For sequencing PCR amplification:
|Screen Says or Select: ||You Press: |
|A: Sandy ||Down arrow, 4
|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 |
|Number of samples ||#
tubes you have, Enter |
|Sample Volume ||your sample volume,
|Lid preheat before auto start||Nothing - you're
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.
Preparing GenBank Submissions with Sequin
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:
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
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.
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:
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.
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 firstname.lastname@example.org. Within a day or two, you should receive an email reply
containing GenBank accession numbers assigned to your temporary taxon codes.
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
TOPO-TA Cloning the Vilgalys-lab Way
- Equilibrate water bath to 42°C.
- Warm vial of SOC to room temperature.
- Warm LBamp, x-gal plates (1 per transformation) at 37°C.
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.
5-30 min at room temperature (maybe 20min optimal?).
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.
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!!
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.
tubes tightly and shake HORIZONTALLY (200 rpm) at 37°C for 1 hour.
all of the cells on a single labeled LBamp, x-gal plate. Incubate overnight at
- Screen white colonies for presence of the desired insert by touching
a colony with a toothpick and "inoculating" a prepared PCR reaction.
screened colonies to a fresh LBamp, x-gal plate, and grow overnight at 37°C. Parafilm
plate edge and refrigerate for long-term storage.
make double-stranded adapters from oligonucleotides:
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.
|Primer ||Core sequence||Enzyme-specific
sequence||Selective base(s) (1 to 3)|
|Purchased by Shian-Ren||Purchased
|EcoRI-GT w/ FAM label (blue) ||EcoRI-C|
unlabeled ||EcoRI-A |
|EcoRI-CA w/TET label (green) ||MseI-A
|EcoRI-CAG unlabeled ||MseI-T|
w/HEX label (yellow) ||MseI-G|
|Tris-HAc pH 7.5||100mM|
Note: a large quantity of this buffer
was made by Shian-Ren. It is stored frozen and is available for use.
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|
|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
II. AFLP ligation - make cocktail for multiple
|volume per reaction||full size||1/4
|10X AFLP Buffer||1µl ||0.25µl |
|T4 DNA ligase||0.25µl||0.06µl|
|5µM d.s. EcoRI adapters||1µl||0.25µl|
d.s. MseI adapters||1µl ||0.25µl |
- 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
III. Preamplification (Optional)
- make cocktail for multiple reactions:
|volume per 25µl reaction|| |
|10X PCR Buffer w/o MgCl2||2.5µl|
|Taq Polymerase||0.1µl |
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|
IV. AFLP amplification:
|volume per 25µl reaction|| |
|10X PCR Buffer w/o MgCl2||2.5µl|
|2mM dNTPs ||2.5µl|
| "EcoRI" primer||2.5.µl|
- Add 24.5µl cocktail to 0.5µl undiluted preamplification
- Amplify according to the following thermocycler specifications:
|File #||File Type||Denaturation
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