LI-COR 4300 User manual

Microsatellite
Analysis
Manual
Model 4300
DNA Analyzer
®

iii

iii
Section 1. Plate Organization
Introduction...................................................................................................1-1
The Hamilton 8-Channel Syringe...................................................................1-1
Microplate Configurations .............................................................................1-1
Section 2. Gel Preparation and Electrophoresis
Choosing Plates and Spacers .........................................................................2-1
Choosing a Gel..............................................................................................2-1
Plate Assembly ..............................................................................................2-2
KB
Plus
Gel Preparation...................................................................................2-6
Preparing Gel Solutions From Other Manufacturers .......................................2-7
Pouring a 6.5% KB
Plus
Gel...........................................................................2-10
Pre-electrophoresis Preparation ...................................................................2-13
Starting Runs ...............................................................................................2-18
Sample Loading...........................................................................................2-18
Disassembly ................................................................................................2-19
Cleanup.......................................................................................................2-20
Section 3. PCR Protocols
dNTP Recommendation ................................................................................3-1
PCR Optimization .........................................................................................3-1
Multiplexing Loci ..........................................................................................3-1
Microsatellite Optimization ...........................................................................3-2
Lab Organization...........................................................................................3-4
Preparing Genomic DNA ..............................................................................3-5
Size Standards ...............................................................................................3-5
Cycling Programs ..........................................................................................3-7
Tailed Primers................................................................................................3-9
Labeled Primers...........................................................................................3-11
Troubleshooting...........................................................................................3-13
Table of Contents

iv
Section 4. Appendices
T
m
Calculation ..............................................................................................4-1
Calculation of Oligonucleotide Concentration (nmol) Given
Optical Density (O.D.)...................................................................................4-2
Protocol for 96-well Paper Combs .................................................................4-3

1-1
Introduction
A system for the arrangement of samples in storage and reaction plates should
be established at the beginning of a microsatellite analysis project. Samples
should be placed in a specific arrangement in the plate to achieve the desired
sample order on the gel. These arrangements can be used for reaction processing
plates as well as stock plates that hold up to a milliliter of the DNA diluted to a
standard concentration.
A consistent volume for each DNA sample can be conveniently transferred to
the 96-well reaction plate with a multichannel pipette or a 96 needle Hydra
(Robbins Scientific). Ideally, samples from an entire family are stored on one
plate and aliquoted to 96-well plates at 20 to 50 ng/µl concentration. Plates are
stored at -20 °C until needed.
The Hamilton 8-Channel Syringe
The Hamilton 8-channel 0.2 mm syringe (Hamilton Co., Part #84511) has
syringe needles are spaced 9 mm apart. Since the wells formed by LI-COR
64-well combs are on 2.25 mm centers, the 8-channel syringe can be used to
load every fourth well of 0.25 mm gels. For 48-well combs, every third lane can
be loaded. LI-COR’s ClickIR Assembly for the Hamilton 8-channel syringe
assures consistent volume delivery through tactile feedback. The ClickIR
assembly attaches to the 8-channel syringe and allows the user to feel clicks that
correspond to 0.3 µl, 0.4 µl, or 0.5 µl (selectable) as sample is dispensed.
Assembly instructions for the ClickIR Assembly can be found in the
Miscellaneous tab of this applications manual.
Microplate Configurations
The arrangement of samples and standards in microplates should be predefined
in order to achieve the desired order on the gel. Two arrangement examples for
64-well combs are shown in Figures 1-1 and 1-2. The wells in each microplate
Plate Organization
1
Section

Section 1
1-2
are numbered according to the lane number that will be loaded. Both
configurations satisfy Saga’s requirement of at least 5 size standard lanes for di-
nucleotide repeats (7 lanes or more are better), as should any plate
configurations you design. The first example shows how to load half of a full
microplate on one gel and the other half on a second gel. The second example
shows how to configure a microplate for loading 60 samples on a 64-well
comb.
Loading 48 Samples on a 64-well Comb
In this configuration, size standards are loaded into every fourth lane. Size
standards (designated "S") are loaded from a second plate or a set of strip tubes.
An entire microplate can therefore be loaded onto two gels.
Figure 1-1. Microplate showing 48 sample loading format. Size standards are loaded
from a second plate or set of strip tubes.
SS SSSSSSSS SSSSSS
14 10713161922
25 11814172023
36 12915182124
25 28 3431 37 40 43 46
26 29 3532 38 41 44 47
27 30 3633 39 42 45 48
®
1 3 7 9 11 13 15 17 21
19 23 25 27 29 31 3533 37 39 41 43 45 4947 51 53 55 57 59 6361
5
1
3
2
26
25
4
5
6
28
29
30
27 33
32
31
9
8
710
11
12
34
35
36 39
38
37
15
14
13 16 19 22
S
SS
S
S
S
SS
2320
18
17
40
41
42 45
44
43
21 24
46
47
48
1
3
2
26
25
4
5
6
28
29
30
27 33
32
31
9
8
710
11
12
34
35
36 39
38
37
15
14
13 16 19 22
23
20
18
17
40
41
42 45
44
43
21 24
46
47
48
1 & 331 & 5
Gel #1
Gel #2
Left syringe tip
starts at well #:
Load #
368
7
6
4
3
2
35
34
4
2
3
Left syringe tip
starts at well #:
Load #
2nd Load
3rd Load
4th Load
1st Load
6th Load
7th Load
8th Load
5th Load

Plate Organization
1-3
Loading 60 Samples on a 64-well Comb
2
1
31
3
4
5
6
7
35
36
32 40
39
11
10
9
812
13
14
15
42
43
47
46
19
18
17
16 20
23 27
S
S
2824
21
22
50
51 55
54
26
25 29
30
58
59
33
34
37
38
41
44
45 49
48 52 56 60
57
53
S
368
7
6
5
4
3
35
34
33
3
4
2
11
2
Left syringe tip
starts at well #:
Load #
S
final load (a single
lane) goes in lane 65
S
9
Figure 1-2. Microplate showing
60 sample loading format.

iii

2-1
Choosing Plates and Spacers
18 and 25 cm gel plates are both suitable for microsatellite analysis
applications. Tri- and tetranucleotide repeats, as well as dinucleotide loci are
clearly resolved on 18 cm gels, providing accurate data. However, for complex
dinucleotide repeats, 25 cm gels provide increased resolution. 18 cm gels
typically resolve fragments of 350 base pairs in 1 hour, while 25 cm gels require
1.5 hours. 0.25 mm spacers should be used with either gel height.
Choosing a Gel
Use 25 cm gels for standard runs since they provide optimum resolution and
have adequate run speeds. Each gel can be loaded up to three times. Throughput
can be increased by using 18 cm gels since they require less time for
electrophoresis between successive loads. 18 cm gels give good performance
with increased speed for many applications.
Table 2-1. Parameters for Standard and Fast Run gels.
Parameter Standard Run Fast Run
Plate Length 25 cm 18 cm
Spacer Thickness 0.25 mm 0.25 mm
Gel Composition 6.5% LI-COR KB
Plus
6.5% LI-COR KB
Plus
Run time (for 350 bases) 1.25 hours 45 minutes
Reload gel after 1.5 hours 1 hour
Gel Preparation and
Electrophoresis
2
Section

Section 2
2-2
Plate Assembly
The following items are required to assemble the electrophoresis apparatus:
• Gloves (non-powdered)
• Safety glasses
• Non-abrasive tissues (Kaydry and Kimwipes)
• Front plate (notched)
• Back plate (rectangular)
• 1 set of spacers
• Comb
• 1 set of rail assemblies
• Casting plate
• Casting stand (optional)
• Concentrated laboratory detergent solution (Micro, Liquinox, etc.
detergent and tap water)
• Deionized water ( ≈ 18.0 MOhm)
• 3-(trimethoxysilyl)propyl methacrylate 98% (bind silane)
• 10% acetic acid
• Test tube or centrifuge tube (must hold 170 µl of liquid)
• Isopropanol (70 - 100%)
Cleaning the Plates
Materials needed:
• 2% laboratory detergent solution made from a concentrate such as Micro
(International Products Corp., Burlington NJ), or Liquinox (Alconox Inc., New
York, NY).
• Spray bottle capable of spraying a fine mist, containing 70% ethanol.
1. Pour a small amount (6 cm circle) of 2% detergent onto the side of the
plate that will contact the gel.
2. Work the solution into a lather with the bristle brush included, and
thoroughly scrub the entire plate. Remove any dried-on poly-
acrylamide with a razor blade, if necessary. Rinse well with water.
Buffer solution that has dried onto the plates can be removed with 1N
NaOH.
3. Pour distilled water onto the plate. Work the water around the plate
with a gloved hand and watch for bubble formation from detergent
residue. Repeat until no bubbles form.
4. Repeat steps 1-3 above with the second plate.
Caution: Always follow proper laboratory safety procedures.
Always wear gloves and safety glasses when working with
chemicals.

Gel Preparation and Electrophoresis
2-3
Preparing Stock Bind Silane Solution
Silane treatments are used in autoradiography to facilitate removal of the gel
from the plates for exposure to film after electrophoresis. Infrared fluorescence
detection does not require repelling silane treatments because DNA bands are
detected in the gel, in real time, during electrophoresis.
3-(trimethoxysilyl)propyl methacrylate (98%), however, is a binding silane used
to covalently bind the gel to the glass in the area where the comb is inserted.
This treatment helps maintain good well morphology when loading gels multi-
ple times.
Applying the Bind Silane Solution
5. Rinse the plates with deionized distilled water. Use a pump spray bottle
to spray a fine mist of 70% ethanol/ddH
2
O for a final rinse. Stand plates
in a rack to air dry.
1. Add 50 µl of bind silane to 10 ml of 100% ethanol.
2. Mix well and store at 4 °C in an amber colored bottle, or wrap the
bottle in aluminum foil.
1. Combine 25 µl of stock bind silane solution and 25 µl of 10% acetic
acid in a 1.5 ml microcentrifuge tube. Mix thoroughly (pipette or
vortex).

Section 2
2-4
Assembling the Gel Sandwich
2. Use a cotton swab to apply the solution on the inside of the short plate
over the area below the edge of the notch where the wells will form
(Figure 2-1). If you are using a squaretooth comb, apply solution to the
rear plate using the front plate as a guide to determine where to place
the bind silane onto the rear plate (other combs do not require back
plate silane treatment). Allow the solution to dry before gel assembly.
Figure 2-1 . Apply bind silane to the plates as shown.
Note that only squaretooth combs require back plate
silane treatment.
1. Lay the rear plate down (gel side up) and place the spacers along the
edges, as shown below.
Figure 2-2 . Place a spacer on the long edges of the plate.
Rear Plate
1.5 cm
Front Plate
Apply bind silane here
Apply bind silane here
Always put the beveled side of the plate to the inside (gel
side). The same side of the plates should always be on the
inside because over time the upper buffer tank gasket
leaves a permanent residue on the plate.
Tip
Rear Plate
Spacers

Gel Preparation and Electrophoresis
2-5
2. Place the front plate on top of the rear plate (gel side down) and align
the spacers with the outside edges of the plates. Make sure that the
plates are aligned evenly at the bottom.
3. Make sure the rails are completely dry from prior runs before assembly.
Place the left and right rail assemblies over the plate edges. Note that
the top portion of each rail is notched for insertion of the upper buffer
tank or casting plate (Figure 2-3). The uppermost clamp knob on each
rail is larger than the other one, as well.
Figure 2-3 . Note orientation of rail assemblies (left rail shown).
4. Check to make sure the rails fit tightly against the edges of both glass
plates (Figure 2-4). The spacer must also be tight against the rail. A leak
will occur if there is a gap between the rail and either plate, or between
the rail and the spacer.
Figure 2-4. Bottom view of assembled gel apparatus
showing the proper fit of the plates and spacer in the rail
Glass clamp knob
Upper buffer tank
clamp knob
Support arm
Groove for upper buffer
tank or casting plate
Notched Front Plate
Rear Plate
Spacer
Rail

Section 2
2-6
KB
Plus
Gel Preparation
Deionized Water Requirements
The conductivity of the water used in the gel and buffer should be 18 MOhm-cm
or greater.
5. Tighten the glass clamp knob on each rail. Tighten only until finger
tight (just past the point of resistance). Over tightening can break or dis-
tort the glass plates. Over tightening is also one of the primary causes of
“smiles” on gel images because distorted plates cause uneven band
migration across the gel.
Assemble the apparatus as shown in Figure 2-5.
Figure 2-5 . Assembled apparatus.
6. Select a comb (sharkstooth or rectangular tooth comb) with thickness
that matches the spacers. Clean plastic combs with water and/or
ethanol if necessary. Make sure that the comb fits between the two
plates at the top of the gel, behind the notch in the front plate. If it
doesn't fit or is very loose, try another comb.
Recheck after tightening all knobs to make sure each knob is
evenly tightened. Also, try to be consistent from day to day
when tightening the knobs.
Tip
Caution: Always follow proper laboratory safety procedures.
Always wear gloves and safety glasses when working with
chemicals.

Gel Preparation and Electrophoresis
2-7
Preparing KB
Plus
Buffer
Gel and running buffer solutions are prepared from a standard 10x TBE buffer.
For best results, KB
Plus
buffer is recommended for use with KB
Plus
gel matrix.
10x TBE Buffer:
Store at room temperature. Note that some precipitation may occur during
prolonged storage.
1X Running Buffer: (Use this buffer for electrophoresis.)
Preparing Ammonium Persulfate Solution
APS provides a source of free radicals needed for polymerization of the gel
(Sambrook, 1989). A 10% APS solution is made by adding 0.1 g APS to 1.0 ml
deionized water in a small test tube. Prepare APS daily to ensure a fresh
working solution. "Stale" APS will adversely affect band quality and give bands a
“fuzzier” appearance with less distinct edges.
Preparing Gel Solutions From Other
Manufacturers
Deionized Water Requirements
The conductivity of the water used in the gel and buffer should be 18 MOhm-cm
or greater.
Preparing Buffer
Gel and running buffer solutions are prepared from a standard 10x TBE buffer.
Gels generally contain 1.0x-1.2x TBE while the running buffer is 0.8x-1.0x TBE.
1. Empty the contents of the KB
Plus
10X TBE pouch into a 1 liter beaker.
2. Add enough distilled water (18M Ω ) to bring volume to about 800 ml.
3. Stir the solution well until all of the solids have gone into solution and
the solution is clear.
4. Add enough water to bring the final volume to 1000 ml.
1. Add 100 ml of 10X TBE prepared as above to 900 ml of distilled water
(18M Ω ).
2. Mix well.

Section 2
2-8
Prepare 10x TBE as follows:
Preparing Ammonium Persulfate Solution
APS provides a source of free radicals needed for polymerization of the gel
(Sambrook, 1989). A 10% APS solution is made by adding 0.1 g APS to 1.0 ml
deionized water in a small test tube. Prepare APS daily to ensure a fresh
working solution. "Stale" APS will adversely affect band quality and give bands a
“fuzzier” appearance with less distinct edges.
1. Add the following to a 1000 ml beaker:
Component Amount Molarity
Tris Base 107.8 g 0.89 M
Boric Acid 55.0 g 0.89 M
EDTA (disodium salt) 7.4 g 0.02 M
Distilled water 950 ml
TOTAL VOLUME 1000 ml
2. Stir to dissolve. Bring to a final volume of 1000 ml.
3. Check the freshly prepared 10x TBE to make sure that it has a pH of 8.3
at 25 °C.
4. Store at room temperature. Note that some precipitation may occur dur-
ing prolonged storage.

Gel Preparation and Electrophoresis
2-9
Acrylamide
The tables below give examples for mixing gel solutions:
Long Ranger™ Gel Components
Page Plus™ Gel Components
A number of other commercial acrylamides can be used to cast gels for the
LI-COR system including RapidGel-XL (USB) and Sequagel (National
Diagnostics).
41 cm 18 or 25 cm
0.25 mm, 6% 0.20 mm, 5.5% 0.25 mm 6.5%
Urea (7M) 12.6 g 12.6 g 8.4 g
50% Long
Ranger™
acrylamide
3.6ml 3.3 ml 2.6 ml
10X TBE
buffer
3.0 ml 3.0 ml 2.0 ml
dd water to 30.0 ml to 30.0 ml to 20 ml
41 cm 18 or 25 cm
0.25 mm, 6% 0.20 mm, 5.5% 0.25 mm 6.5%
Urea (7M) 12.6 g 12.6 g 8.4 g
40% Page
Plus™ acryla-
mide
4.5 ml 4.125 ml 3.25 ml
10X TBE
buffer
3.0 ml 3.0 ml 2.0 ml
dd water to 30.0 ml to 30.0 ml to 20 ml
To mix the gel solution in one beaker: Place a 100 ml beaker with a
stir bar on a balance and tare the balance. Measure 12.6 grams of
urea into the beaker, then add the acrylamide solution and 10x TBE
buffer. Add dd water to target volume of 30 ml gel solution).
Tip

Section 2
2-10
Mixing the Gel Solution
Pouring a 6.5% KB
Plus
Gel
The following items are required to pour the gel:
• 20 ml of 6.5% KB
Plus
Gel Matrix for Genotyping
•KB
Plus
1X TBE buffer (recommended for use with KB
Plus
gel matrix)
• 150 µl of 10% Ammonium persulfate (APS)
• Comb
• 60 cc syringe with 14 gauge tip
• Pasteur pipette
• Assembled gel sandwich and casting stand (optional)
• 15 µl of TEMED
1. Mix well at room temperature and filter the solution using a membrane
filtration system (syringe top or bottle top from any supplier).
2. Add 150 µl of 10% APS per 20 ml of gel solution and swirl gently.
3. Just before pouring, add 15 µl TEMED. You have approximately 3-5
minutes to pour the gel before it polymerizes.
1. Bring 20 ml of KB
Plus
to room temperature (10-15 minutes) and prepare
glass plates for gel injection while KB
Plus
warms.
2. Add 150 µl of 10% APS and 15 µl TEMED when ready to inject gel
solution.
3. Mix to homogenate and draw the gel solution into a 60 cc syringe with
14 gauge tip.

Gel Preparation and Electrophoresis
2-11
4. For 25 cm gels, a notch on the back of each rail that allows the appara-
tus to rest on the uppermost metal posts on the casting stand (Figure 2-6).
This slight incline improves the flow of the gel between the plates.
Figure 2-6. Rest the apparatus on the casting stand (25 cm plates).
Start a little above the bottom of the notch at the left or right side of the
notch in the front plate. Inject the gel evenly at a steady rate while mov-
ing downward to the bottom of the notch and then side to side across
the notch. Periodically tap the front of the plates firmly to prevent the
formation of air bubbles. If the gel is being injected correctly, you
should get a smooth half moon shaped gel front advancing downward
between the gel plates. If plates are dirty, the advancing primer front
will be jagged. Never pull up the syringe after you start injecting. Any
time you stop you are likely to create an air bubble. When the gel solu-
tion reaches the bottom of the plates and a small pool of gel overflows
onto the notch in the front plate, quickly lay the plate assembly flat on
the bench to prevent the gel solution from running out the bottom.
5. Remove any bubbles that form during gel pouring using a bubble hook
(Figure 2-7).
Figure 2-7 . Bubble Hooks.
Yellow: 0.25 mm diameter
Blue: 0.20 mm diameter

Section 2
2-12
6. Insert the comb. Figures 2-8 and 2-9 show how to insert the mylar
sharkstooth and rectangular tooth combs after pouring the gel. Instruc-
tions for inserting paper combs are given in the Appendices (Section 5).
The sharkstooth comb is inserted upside down during polymerization to
make a trough which forms the base of the wells, and is then inverted
before loading the samples.
Figure 2-8 . Center the comb in the notch and insert the
sharkstooth comb upside down until the plastic depth
gauge rests on top of the notch.
Figure 2-9 . Center the comb in the notch and insert the
rectangular tooth comb with the teeth downward, until the
plastic depth gauge rests on the notch.
Insert the comb slowly to avoid air bubbles forming around the comb.
Air bubbles can destroy or deform the wells. Add a small amount of the
gel solution over the comb (near the notch) to compensate for gel
shrinkage as it polymerizes.