Appendix 1
***All methodologies and explanations were
provided by Dr. Travis Glenn (Univ. of South Carolina and Savannah River
Ecology Lab). All text and subsequent errors are the efforts of Ryan Thum
(Dartmouth College- Dept. of Biological Sciences) and Pam Svete (Univ. of
Alaska Fairbanks- Dept. of Biology and Wildlife).
METHODS ASSOCIATED WITH MICROSATELLITES:
General Steps: MINIMAL!!!
estimated time:
(This, for the
most part, is how far I was able to get in @ 3 months with an established lab
and
a fair amount of
previous microsatellite experience.)
21. Label primers with fluorocein markers 2 days
22. Reoptimize
PCR parameters for primers 2-7
days
23. Optimize primers on automated
sequencer 2-4
weeks
24. Extract DNA 1-3
weeks
25. Conduct microsatellite analysis with
automated sequencer 1-2
months
26. Statistical analysis of microsatellite
data 1-2
months
Isolate DNA
Notes: Various types of extraction methods
are possible. I prefer Qiagen when I can afford it. Qiagen kits are
useful for the extraction of multiple tissue types (pg 19 of Qiagen Tissue
Extraction kit. As well, Qiagen has 96-well extraction kits called "Dneasy
96 Tissue Kit # 69581. To order Quiagen kits call 1800-426-8157). This lab also
uses PCI extractions and mud extractions.
General Comments:
There are many strategies for obtaining total
genomic DNA from various organisms of interest. To develop a microsatellite
library, it is only imperative that you have one individual. It is best to use
the heterogametic sex in diploid, sexual organisms in the event that
sex-specific markers exist.
When extracting DNA from an organism for the
first time it sometimes helps to try a few protocols and see which one works
the best for you in terms of time spent in extraction, cost, and safety
concerns.
Two protocols have been listed below that have
been quite successful across a wide range of animal taxa. There are numerous
protocols for DNA extraction and all are easily obtainable. In addition, kits
can be purchased to extract DNA and work very well despite the expense
PCI extraction works very well and is easy to
perform, however, disposal of waste can be cumbersome and the chemical reagents
are very toxic. PCI extractions should always be performed underneath a fume
hood with protective clothing, protective eyewear, and gloves.
MUD extraction is simple and cheap. In many
cases, the initial THE-Proteinase K digest can be skipped. Alternatively, the
organismal sample can be digested at length in the Guanidinium extraction
buffer without prior Pro-K digestion.
Following DNA extraction, it is best to quantify
the recovery of total genomic DNA obtained from the organism of interest. There
are a few different strategies employable to achieve DNA quantification.
Spectrophotometers and Fluorometers are available from a variety of
manufacturers. Perhaps the simplest way to determine the success of a DNA
extraction is to run the sample on an agarose gel. This method is perfectly
acceptable for microsatellite development purposes.
To quantify DNA, make a 1.5% agarose gel. Run
out samples next to some known size and quantity standards. Various quantities
of uncut lambda (L) DNA can be run out for quantification estimates. In
addition, a size standard ladder such as Hi-Lo Marker can be run out to check
for DNA fragment size distributions. If staining samples with SYBR Gold stain,
it is best to stain in a bath in order to avoid biasing size estimates. If
using Ethidium Bromide, it can be placed directly in the agarose gel before
pouring.
Guanidinium thiocyanate recipe:
Mix the following:
96 g GuSCN
82.56 mL dH2O
8 mL 1M Tris (pH 8)
7.04 mL 0.5M EDTA
2 mL Triton x -100
**Insert the PCI and MUD protocols
A. MUD DNA Extraction
Solutions:
Diatomaceous Earth (MUD when wet)-
Add
10 mL of water to 50 mL graduated cylinder
Add
approximately 5 g of DE using a funnel
Fill
graduated cylinder to 50 mL with diH2O
Cover
cylinder with plastic and resuspend DE into water
Allow
diatoms to settle for 3 + hours
Remove
upper liquid layer
Refill
to 50 mL with diH2O and mix
Transfer
to 50 mL conical and let settle for 2 + hours
Remove
upper liquid layer
Add
a volume of diH2O equal to the MUD
Vortex
vigorously immediately before using
Extraction Buffer-
Combine
24 g GuSCN and 22.7 mL (= 0.5 g) MOPSO (or 20 mL 0.1M Tris-HCl pH 6.4) in a 50
mL conical
Heat
to 60o to dissolve the GuSCN
Add
0.5 mL Triton X-100 (or SDS, or Tween-20)
Add
1.76 mL of 0.5M EDTA
Fill
to 50 mL with diH2O
Washing Ethanol-
70%
Ethanol supplemented with 10mM NaCl (optional). This works the fastest if it's
in a squirt bottle.
1) Add a "goober" (standard IU
measurement unit for small amount) of frozen (or liquid) blood to approximately
1 mL of GuSCN extraction buffer.
2) Incubate on rotator (AKA spinny twirly
thingy) at 55o for anywhere from 2 hours to overnight (until there are no gobs
in the solution).
3) Add 75 _L of MUD.
4) Incubate on rotator at 55o for at least
ten minutes and up to several hours.
5) Vortex briefly, centrifuge briefly, and
discard supernatant.
6) Add approximately 1 mL of 70% ethanol
(preferably cold) to the mud pellet, vortex briefly, centrifuge for 2 minutes,
and discard the ethanol supernatant.
7) Repeat step 6. Dry the MUD pellet either
using low heat or at room temperature or under the fume hood.
8) Add 125 _L of TE to the mud pellet.
9) Vortex briefly to break up the pellet and
incubate on rotator at 55o for up to several hours.
10) Vortex briefly, centrifuge for 2 minutes.
11) Remove supernatant containing DNA
suspended in TE (...theoretically at this point) and place in microcentrifuge
tube. Can repeat steps 8 - 11 for a second aliquot if you like.
12) Save mud pellets in 20o freezer.
B. Quantification of DNA
At this point, it is recommended to stain the
gel after running the samples so to avoid molecular weight bias by the
sybergold or ethidium bromide. 1% agarose gels are fine for DNA quantification.
A 1.5% gel may be preferred for higher resolution if the fragment is 500bp or
less. Overall, the higher % agarose in a gel will yield better separation of
fragments. $- Agarose costs @ $1 per gram.
1% Agarose Minigel:
In a 200ml glass erlenmyer flask, mix the
following:
0.35 g agarose
35 ml 1X TBE
- Microwave for about 1 minute. Let solution
cool to 55ƒ C. Pour into tray sealed with tape, add comb, and let
agarose set (will turn cloudy).
- DNA can be mixed with dye on parafilm while
loading gel:
5
m L DNA + 2 m L blue dye
- For the standard mix:
2m L HiLo Marker
+ 2 m L blue dye
1.5% "Centipede Gel": (1 comb = 50 wells)
Mix the following:
3.0 g agarose
200 ml of 0.5X TBE
Gel photo at USC:
-Check gel on lightbox in lab
-Go to room 704A (code to enter ####)
-Open photo machine, turn on the "epi
white" light
-Clean the black surface
-Go to computer and open the program
"alpha mager 2000"
Login:
name "****", password "****"
-Position gel on the black surface in the
photo machine
-Zoom and focus
-Turn off "epi white" light and
close photo machine
-Turn on photo machine by pressing
"power"
-Go to computer and click "expose,"
use the right arrow under the exposure time window to make it faster
-When the photo is satisfactorily exposed
click "freeze"
-Click "toolbox 2" and under
"filters" select "sharpen media"
-Click on the icon for text "A" to
label the photo or specific lanes
-Save photo to disk
Cut genome with restriction enzyme
After extraction and quantification of
isolated DNA products, the genome should be cut into smaller fragments before
attempting to go any further with microsatellite development. A variety of
restriction endonucleases can be utilized effectively to achieve this end. Two
factors however are extremely important in deciding which enzyme to use. The
first factor is which enzyme(s) will be compatible with the linker or adapter
that will be employed in subsequent steps if using ligated linkers for PCR of
fragments throughout the development process. The second factor is the desired
average size of genomic fragments. It is recommended to use restriction enzymes
that will result in an average fragment length of 500 base pairs (hereafter
bp). Enzymes such as Dpn II or Mbo I will easily accomplish this
for most genomes.
The following protocol uses Dpn II
restriction digest as a model for this step of the microsatellite development
process. It is important however to note that this protocol can be modified to
fit the developer’s needs for specific applications. In addition, enzyme reaction
conditions should be carried out according to the manufacturer’s
specifications. This protocol assumes a 50 µL reaction volume, however, the
various reagent volumes can be modified as well.
Notes: Dpn2 consists of 4 bp which will cut
approximately every 256bp. Optimally, one should only cut with Dpn2 for one
hour.
Dpn2 cleaves at the beginning of GATC.
x µL of organismal genomic DNA (~2,500-3,000
ng total genomic DNA)
5
µL of 10x buffer for Dpn II (1x final conc.)
2
µL of Dpn II (use more or less depending upon final volume)
*
x µL of diH2O to bring final volume up to 50 µL
* Depending upon the concentration of the
genomic DNA to be cut, the reaction volume can be more or less than 50 µL.
2) Incubate at 37o for one hour.
3) Terminate reaction by heating to 70o for
20 minutes. Alternatively, 5 µL of EDTA may be added to the mixture to
terminate the reaction, however, this should be followed by subsequent PEG
purification before proceeding to the next step.
NOTE: Restriction enzyme should be kept on ice until use and should be
placed immediately back in -20o freezer.
Ligate linkers to DNA fragments
Note: Though not described in full until
later. A PEG procedure should proceed this step.
Ligating linkers onto the genomic DNA
fragments has several advantages. The linkers provide a priming region for all
subsequent PCRs. In addition, if molecular cloning is to be employed in the
microsatellite development process, the linkers provide a cloning site for
ligation into specific vectors. It is thus important to use linkers that are
compatible with both the restriction enzyme used for genomic digestion and the
vector cloning site to be utilized.
This step, as well as the genomic restriction
endonuclease digest can be foregone if DOP-PCR is used to amplify the genome
according to Koblizkova et al. (1998). The basic concept however remains the
same: before enrichment of microsatellite-containing fragments within the
organismal genome, all fragments must contain a primable sequence on either end
for amplification and subsequent sequencing.
The following protocol uses the Sau L linkers
A and B and a 25 µL reaction as a model for the process of providing a common,
primable sequence of nucleotides on the of each genomic fragment.
1) Mix the following in a microcentrifuge
tube:
1 µg of ~500 bp
fragments (equivalent to ~ 6.6 pmol of fragment ends)
2.5 µL 10X DNA ligase buffer (1x final
concentration)
3-5 times the number of linker pmols as
fragment pmols
1 µL of DNA ligase
bring up to final volume of 25 µL with water
NOTE: Restriction enzyme should be kept on ice until use and should be
placed immediately back in -20o freezer.
** It is also useful to run plasmid, cut
plasmid, and cut plasmid treated with calf-intestinal alkaline phosphatase
(CIAP) on the same gel.
COMMENTS: Again, the volumes are not important in this protocol.
Rather, the relative amounts of fragment ends to linker ends and the 1x buffer
concentration are most important. Three to five times the amount of linker ends
relative to DNA fragment ends is desired. This will put the odds of ligating
linkers to each fragment in your favor over ligating two genomic DNA fragments
together.
HELPFUL CONVERSIONS:
1 µg of 1,000 bp fragments = 3.3 pmol
fragment ends
after restriction digest, the average
fragment length is ~500 bp, therefore, 1 µg of 500 bp fragment ends = 6.6 pmol
of ends.
1 µL of x µM linkers = x pmol of linker ends
QUICK AND EASY DETERMINATION OF LINKER
AMOUNTS:
Add 1 µg of fragments (equivalent to 6.6 pmol
of fragment ends)
Multiply fragment pmol by 5 = ~ 35 pmol
Substitute into equation: (1 µL)(x µM
linkers) = x pmol linkers
PEG
Notes: Here, this step serves the purpose of
getting rid of extra linkers.
PEG Precipitation of PCR Products For a
50 _L PCR Reaction
Solutions:
20% PEG, 2.5M NaCl- for 50 mL conical, mix:
10
g PolyEthylene Glycol (MW 6000 - 8000)
7.3
g NaCl
fill
to 45 mL with diH2O- shake and put in rocking incubator at 37o to let PEG go
into solution
fill
to 50 mL after everything is in solution
80% ethanol
TLE- 10mM Tris, 0.1 mM EDTA
1) Add 50 _L of PEG (equal volume) to a 0.5
mL reaction tube. Transfer the PCR product to the reaction tube with PEG and
mix by pipetting up and down (AKA "sqlooshing).
2) Let this incubate for 15 minutes at 37o.
Place a bottle of 80% ethanol on ice while the PEG mixture is incubating.
3) Centrifuge the mixture at high speed for
15 minutes at room temperature.
4) Pull off the supernatant and discard using
a pipetter.
5) Add 125 _L (2.5 to 3 volumes) of ice-cold
ethanol. It is best to spin for 2 minutes, however, if you didn't drop the
ethanol into the bottom of the tube (but dripped it down the side), you can
just let it sit for a minute.
6) Pull off the ethanol and discard in the
waste ethanol container.
7) Repeat the ethanol wash.
8) Dry the remaining ethanol using a
centrivap at low heat for a few minutes. There should be no signs of ethanol
(visual or olfactory) when done.
9) Dissolve pelleted PCR product in 25 _L of
TLE. Sqloosh to make sure the pellet goes into solution and it is best to let
it sit for a little while before using.
10) Run a gel for ~10 minutes to quantify recovery
using 20 or 100 ng of uncut lambda or plasmid DNA as a standard.
PCR
Notes: This procedure serves to make lots of
template for enrichments.
PCR of Linker Ligated DNA Fragments
Using Sau L A Primer
For 50µL reaction:
5 µL 10X
PCR reaction buffer
2.5 µL 10µM
Sau L A primer (Linker A)
3 µL dNTP's
4 µL MgCl2
(25 mM)
32.9 µL H2O
0.1 µL Taq
DNA polymerase
2.5 µL template
DNA fragments
NOTE: template DNA may be increased or
decreased and compensated for with water.
Cycling:
Initial denaturation: 94m for 2 minutes
25 Cycles:
94ƒ for 30 seconds
50ƒ for 30 seconds
72ƒ for 1 minute
Hold at 15ƒ
Enrich (for specific repeat)
Note: Probes may be made in the following
manner or purchased.
A. Preparation of Oligonucleotide Probes
for use in DNA Hybridizations using Boehringer Mannheim 3'-End Labeling Kit
1) Mix the following reagents on ice:
4
m L tailing buffer (vial 1)
4
m L CoCl2 solution (vial 2)
100
pmol of oligonucleotide (use following: 1 m L * x microM DNA =
x pmol)
1
m L DIG-ddUTP solution (vial 3)
1
m L (=50 units) terminal transferase (vial 4)
2) Incubate at 37ƒ for 15 min, then place on ice again.
3) Add 1 m L of glycogen
solution (vial 8).
4) Precipitate the labeled oligonucleotide
with 2.5 m L of 3M sodium acetate pH 5.2 and 75 m L of ice-cold 95% ethanol.
5) Place in -70ƒ freezer
for at least 30 minutes or -20o freezer for at least 2 hours.
6) Centrifuge (at 12,000 g), wash the pellet
with 50m L ice-cold 70% ethanol. Dry using centrivap at 60ƒ . Redissolve pellet in 50 m L TE.
7) Store labeled probe in -20ƒ freezer if not being used immediately.
B. Biotin-Avidin Bead Enrichment
Procedure for Microsatellite-containing Genomic DNA
1) Denature 50 µL of cut, linker-ligated
genomic DNA at 95ƒ for 5 minutes.
2) Add denatured DNA to 340 µL of
hybridization buffer and 10 µL of biotinylated microsatellite probe.
3) Incubate on rotator at 55ƒ for at least 30 minutes to overnight.
4) *Split mix into two 200µL aliquots and add
25 µL of Avidin D beads to each mix.
5) Incubate on rotator at 55ƒ for one hour.
6) Spin mix at 15,000x for a couple of
minutes and remove hybridization buffer supernatant. Save supernatant for
troubleshooting purposes.
7) **Wash Avidin beads several times with 2X
SSC, each time spinning down, removing the supernatant and saving it. More
stringent washes can be used successively by decreasing the concentration of
SSC in the wash if desired. It is recommended however that you control for the
stringency in the other aliquot by washing an equal number of times with only
2X SSC.
8) Elute theoretically
microsatellite-enriched DNA using 100 µL of 200 mM NaOH. Spin on rotator at 37_
for approximately 15 minutes.
9) Spin down for a few minutes and remove
supernatant. Neutralize supernatant with .36 µL of 11.6 Molar HCl. Add 10 µL of
Tris pH 8.0 to buffer neutralization.
10) Perform PCR on supernatant to use for
ligation into vector.
* The mixes are separated to wash with
different stringency regimes if desired.
** More stringent washes may increase the
relative proportion of long microsatellite sequences in cloned colonies,
however, they may also cause the loss of microsatellite sequences via undesired
elution during washing.
Cut off linkers with BamHl
Notes: BamHI will cut in the middle of the
linker
1. Label 1.5 L m vials Ex.
"MDK T0 .1X Dpn2"
X 1
DNA 15m L
DpnII
buffer 5 m L
DpnII 2 m L
H20 28 m L
PEG
PEG Precipitation of PCR Products For a
50 m L PCR Reaction
Solutions:
20% PEG, 2.5M NaCl- for 50 mL conical, mix:
10
g PolyEthylene Glycol (MW 6000 - 8000)
7.3
g NaCl
fill
to 45 mL with diH2O- shake and put in rocking incubator at 37o to let PEG go
into solution
fill
to 50 mL after everything is in solution
80% ethanol
TLE- 10mM Tris, 0.1 mM EDTA
1) Add 50 _L of PEG (equal volume) to a 0.5
mL reaction tube. Transfer the PCR product to the reaction tube with PEG and
mix by pipetting up and down (AKA "sqlooshing).
2) Let this incubate for 15 minutes at 37o.
Place a bottle of 80% ethanol on ice while the PEG mixture is incubating.
3) Centrifuge the mixture at high speed for
15 minutes at room temperature.
4) Pull off the supernatant and discard using
a pipetter.
5) Add 125 _L (2.5 to 3 volumes) of ice-cold
ethanol. It is best to spin for 2 minutes, however, if you didn't drop the
ethanol into the bottom of the tube (but dripped it down the side), you can
just let it sit for a minute.
6) Pull off the ethanol and discard in the
waste ethanol container.
7) Repeat the ethanol wash.
8) Dry the remaining ethanol using a
centrivap at low heat for a few minutes. There should be no signs of ethanol
(visual or olfactory) when done.
9) Dissolve pelleted PCR product in 25 _L of
TLE. Sqloosh to make sure the pellet goes into solution and it is best to let
it sit for a little while before using.
10) Run a gel for ~10 minutes to quantify recovery
using 20 or 100 ng of uncut lambda or plasmid DNA as a standard.
Ligate into plasmid
Notes: ligate via the restriction sites
When making master mix, add ligase at the end
because it is very sensitive
-Gather supplies and label vials
-Make Master Mix
X 1
PCR
product 3
m L
Puc18 BamHI/BAP vector 1 m L
Buffer 1
m L
Ligase 1
m L
H20 4
m L
-Soak 17ƒ C for 1 hour
(you can use an old thermal cycler for this)
Transform bacteria
A. Making agar plates:
Agar Solution:
25 g LB Broth
15
g agar
1000
ml H2O
Autoclave
Cool
to 50ƒ
B. TRANSFORMATION PROTOCOL from Stratagene
1. Thaw two tubes
of SoloPack Gold cells on ice (one tube is for experimental transformation and
one tube is for the control transformation)
2. When the cells
have thawed, swirl the tube to gently mix the cells
3. Add 1 m L of of the XL10-Gold b -mercaptoethanol misx provided
in the kit to each tube [Stratagene cannot guarantee the highest efficiency
with b -ME from other sources (see Use of b -Mercaptoethanol)].
4. Swirl the tube
gently. Incubate the cells on ice for 10 minutes swirling the tube every 2
minutes.
5. Add 0.01- 50 ng
of DNA to one of the tubes of cells and swirl the tube gently. [Although we
ignore this part, Stratagene suggests: As a control add 1 m L of the pUC18 DNA control plasmid (diluted 1:10 in high quality
water) to the other tube of the cells and swirl the tube gently.]
6. Incubate the tubes on ice for
30 minutes.
7. Heat-pulse the
tubes of SoloPack Gold competent cells in a 42ƒ C water
bath for 60 seconds. (Temperature and duration of heat-pulse are critical for
obtaining the highest transformation efficiency.)
8. Incubate the
tubes on ice for 2 minutes.
9. To each tube of
SoloPack Gold competent cells, add 0.175 ml of NZY+ broth and incubate the
tubes at 37ƒ C for 1 hour with shiaking at 225-250 rpm.
10. Plate 100 m L of the experimental transformation reaction on LB-ampicillin agar
plates using a sterile spreader.
11. Incubate the
plates overnight (12-16 hours) at 37ƒ C.
Final Note: For
SoloPack Gold competent cells, for 5m L of transformation
plated, 25 colonies can be expected with an efficiency of > 1X108 cfu/m g of pUC18 DNA.
-(Blue white selection)
Notes: This part of the protocol has not been
working
PCR
Notes: When plates of PCR reactions are run,
primer is decreased for the purpose of possibly avoiding PEG.
PCR of Positive Screened Colonies
Following Transformation
For a 25 m L reaction:
1
plate 2 plates
X
1 X 96 X 192
(m L) (m L) (m L)
250 m g/ml BSA (Bovine
Serum Albumin) 2.5 275 550
10X PCR reaction buffer 2.5 275 550
10 m M M13 forward
primer .625 68.75 68.75
10 m M M13 reverse
primer .625 68.75 68.75
50 mM MgCl2 0.75 82.5 165
25 mM dNTP’s 1.5 165 330
H20 10.25 1265.0 2678.5
Taq DNA Polymerase 0.1 11 11
DNA template from bacteria colony suspended
in 100 m L H20 5.0 5.0 5.0
NOTE: template DNA may be increased or
decreased and compensated for with water.
Cycling:
Initial denaturation: 94ƒ for 3 minutes
35 Cycles:
95ƒ for 20 seconds
50ƒ for 20 seconds
72ƒ for 1 minute 30 seconds
Hold at 15ƒ
Dot Blot
A. Hybridization of Oligonucleotide
Probes to Genomic DNA using Boehringer Mannheim Kit
Solutions:
Prehyb/Hyb Buffer- For 1 Liter mix
250
mL 20X SSC
10
mL 10%
N-Lauroylsarcosine
10
mL blocking buffer
(10% blocking reagent diluted 1:10)
2
mL 10% SDS
Bring
up final volume to 1 Liter.
B. Immunological Detection (AKA
"Dot Blots") for Hybridization of Oligonucleotide Probe to Genomic
DNA using Boehringer Mannheim Kit
Solutions:
Buffer 1 (Maleic Acid buffer)
0.10 M Maleic Acid
0.15 M NaCl
pH 7.5 (Adjust
using solid or concentrated NaOH)
Autoclave
OR for 1 Liter of
Buffer add
11.607g Maleic Acid
8.766g NaCl
pH 7.5 (40 pellets
of NaOH, then drops)
Washing Buffer
Buffer 1 with Tween
20, 0.3% (W/V)
Buffer 2 (Blocking buffer)
Blocking readgen
diluted 1:10 in Maleic acid buffer (final concentration is 1% blocking reagent)
Buffer 3
100mM Tris-HCl
100 mM NaCl
50 mM MgCl2
pH 9.5
OR for 1 Liter of
Buffer add
0.51g MgCl2
0.29g NaCl
0.1 M Tris pH 9.5
added to make final volume 50 ml
Buffer 4 - TE
10
mM Tris-HCl
1 mM EDTA
pH 8.0
Cycle sequence (forward and reverse)
Note: At SREL, solutions for Cycle sequencing
reside in the -20ƒ in a box labeled "Susan take to SREL Fri Feb
26". The cocktail vial may be reused.
At SREL the T.C. program is #25
At USC the T.C. program is #01
Combine the following:
X 1
(m L)
Big Dye Terminator mix 4.0
H2O 3.0
DNA template 2.0
(*Forward or Reverse accordingly)
Thermal Cycler Settings:
1 cycle:
94ƒ 3 min.
25 cycles:
96ƒ 5 sec.
50ƒ 5 sec.
60ƒ 4 min.
15ƒ hold
Automated Sequencing
A. Labeling cycle sequences for the
automated sequencer.
My current understanding is that Lynette will
be running sequences on a regular basis at USC every Thursday. The following
labeling description is based on my personal experience with loading gels. Gels
must be loaded backward (lane 48 to 01) because the comb fits more
"loosely" near lane one; and loaded every other sample to avoid
sequence contamination from the wells "leaking". It can get
confusing.
In conjunction with Msat development:
Following the Dot Blot, I draw a table in my
notebook (1-12 X A-H). Then I will write Light, Medium, Dark (L, M, D) in the
corresponding blank of the table. This provides a template by which I know
where the msat rich PCR product is located on the 96-well plate.
Example:
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
A |
|
|
|
|
|
|
L |
|
|
|
|
|
|
B |
|
|
|
D |
|
|
|
M |
|
|
L |
|
|
C |
|
|
L |
D |
|
|
|
|
|
|
|
|
|
D |
L |
|
M |
|
|
|
|
|
|
|
|
D |
|
E |
M |
D |
|
|
|
|
|
|
|
|
|
|
|
F |
|
|
|
|
|
|
|
|
|
|
|
|
|
G |
|
|
|
|
L |
M |
|
|
|
|
|
|
|
H |
|
M |
|
|
M |
|
|
|
|
|
|
|
Next, I name each of these samples using the
following nomenclature:
C-T2-1- ‘X’
C
= species type, Chipmunks here
T2
= probe type used during enrichment. T0, T2, and T3 probe
for tetramer repeats. HT and LT probe trinucleotide repeats, and AG
probes for dinucleotide repeats.
1
= plating order. When plating cells post-transformation, only 100 m L of the cells are used. Thus, ‘2’ would correspond to the second
plating of this tube of transformed cells. This number is important to remind
you to check if the same microsatellites were transformed more than once, and
to avoid duplicating efforts when designing primers.
X
= the sequential number of the specific msat. which defines each locus uniquely
When I cycle sequence, it is important to
consolidate information on the strip tubes because the writing area is quite
limited. Thus, I define the strip tube by the page in my notebook on which the
important information is written (‘PS 48’); I write this on the back of
the tube. On the ends of the strip, I write the number of the strip relative to
the group (‘01’). Then on the upper lip of each tube (the part which is
never directly exposed to heat), I label each tube ‘a’ through ‘h’.
Thus, when I am loading samples, I have a quick reference (b/c I am checking
off a copy of the sample sheet as I load) to ensure that the correct sample is
going into the corresponding lane.
Example of notebook:
(Columns:
PS 48
|
1d |
C- |
T0- |
1- |
01 |
PS48- |
01- |
A |
|
1e |
|
|
|
02 |
|
|
B |
|
2e |
|
|
|
03 |
|
|
C |
|
2h |
|
|
|
04 |
|
|
D |
|
3c |
|
|
|
05 |
|
|
E |
|
3d |
|
|
|
06 |
|
|
F |
|
4b |
|
|
|
07 |
|
|
G |
|
4c |
|
|
|
08 |
|
|
H |
|
5g |
|
|
|
09 |
|
|
A |
|
5h |
|
|
|
10 |
|
|
B |
|
6g |
|
|
|
11 |
|
|
C |
|
7a |
|
|
|
12 |
|
|
D |
|
8b |
|
|
|
13 |
|
|
E |
|
11b |
|
|
|
14 |
|
|
F |
|
|