***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:

1. Isolate DNA 1 day
2. Chop DNA with Dpn2 1 hour
3. Linkers 2 hours
4. PEG 2 hours
5. PCR (make lots for enrichments) 4 hours
6. Enrich (for specific repeat) 3 hours
7. Cut off linkers with BamHl 1 hour
8. PEG 2 hours
9. Ligate into plasmid via the restriction sites 2 hours
10. Transform bacteria 2 hours+ 16 hour incubation
11. (Blue white selection)
12. PCR 4 hours
13. Dot Blot 1 day
14. Cycle sequence (forward) 3 hours
15. Sequence (ABI 377 Automated Sequencer)
16. Check sequences for msats. with Sequencher software 2 days
17. Cycle sequence (reverse) 3 hours
18. Design and order primers 1-2 days
19. Optimize primers 1-2 weeks

(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.)

20. Determine if primers variable 1-2 weeks

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.

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.

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

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.

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.

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 55° 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

• Run gel for a minimun of 15 minutes at @ 86 volts.

1.5% "Centipede Gel": (1 comb = 50 wells)

Mix the following:

3.0 g agarose

200 ml of 0.5X TBE

-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

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.

1. Mix the following in a microcentrifuge tube:

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 37^o for one hour.

3) Terminate reaction by heating to 70^o 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 -20^o freezer.

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:

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

2. Incubate at 17º for a few hours to a few days.

NOTE: Restriction enzyme should be kept on ice until use and should be placed immediately back in -20^o 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.

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

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

Notes: Here, this step serves the purpose of getting rid of extra linkers.

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.

Notes: This procedure serves to make lots of template for enrichments.

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:

94° for 30 seconds

50° for 30 seconds

72° for 1 minute

Hold at 15°

Note: Probes may be made in the following manner or purchased.

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 37° 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 -70° 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 60° . Redissolve pellet in 50 m L TE.

7) Store labeled probe in -20° freezer if not being used immediately.

8. Detect amount of labeled probe using B-H detection protocol.

1) Denature 50 µL of cut, linker-ligated genomic DNA at 95° 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 55° 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 55° 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.

Notes: BamHI will cut in the middle of the linker

1. Label 1.5 L m vials Ex. "MDK T0 .1X Dpn2"

2. Add 15 m L PCR product to each vial
3. Make cocktail for cutting linkers

X 1

DNA 15m L

DpnII buffer 5 m L

DpnII 2 m L

H20 28 m L

4. Incubate at 37° C for 1 hour (do not shake).

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.

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

1. label vial as "B1 To 2X PUC"
2. will need:

1. PCR product
2. puc18 BamHI/ BAP vector (Amersham Pharmancia Biotech Inc. 27-4855-01)
3. NE Buffer for T4 DNA Ligase with 10mM ATP (by Biolabs)
4. H2O
5. Ligase T4 DNA Ligase (by Biolabs #202)

-Make Master Mix

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 17° C for 1 hour (you can use an old thermal cycler for this)

15 g agar

1000 ml H2O

Autoclave

Cool to 50°

1. Make agar
2. Stir with stirbar
3. Add Ampicilian so final concentration is 75 m g/ml.
4. Add Tetracyclin so final concentration is 10 m g/ml.
5. Pour onto bacterial plates (about halfway plate height, approximately 20 ml per plate).
6. Avoid bubbles.

Notes: This part of the protocol has not been working

Notes: When plates of PCR reactions are run, primer is decreased for the purpose of possibly avoiding PEG.

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: 94° for 3 minutes

35 Cycles:

95° for 20 seconds

50° for 20 seconds

72° for 1 minute 30 seconds

Hold at 15°

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.

1. Add appropriate amoung to Hyb solution to filter paper with cross-linked denatured DNA (5m L DNA, 5 m L 1 M NaOH or formamide at 95° for 1-2 minutes; 2 m L dot on nylon filter paper cross-linked using UV light for 5 sec (with new machine at SREL. May UV for 1 minute with different machine).
2. Shake at 55° for approximately 15 min. to 1 hour. Then pour off Hyb solution.
3. Add Hyb soln again with added probe (15m L) and shake again for one hour at 55° .
4. Remove Hyb soln, but save and freeze b/c can be reused.
5. Let filter paper and container cool to room temperature.
6. Wash with 2X SSC/ 0.1% SDS at 40° long enough that the solution reaches 40° .
7. Repeat step 6
8. Wash with .2X SSC/ 0.1% SDS at room temperature
9. Repeat step 8
10. Detect using Boehringer Mannheim immunological detection protocol

Solutions:

1. M Maleic Acid
1. 0.15 M NaCl

pH 7.5 (Adjust using solid or concentrated NaOH)

Autoclave

OR 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 0.51 MgCl2

1. M NaCl/ 0.1 M Tris pH 9.5 added to make final volume 50 ml

10 mM Tris-HCl

1. mM EDTA

1. Wash filters briefly (approximately 1 minute) in buffer 1 + Tween 20, 0.3% (w/v)
2. Place in Buffer 2 (witch is located in the refrigerator) and incubate at room temperature for about 10- 30 minutes
3. Pour off Buffer 2
4. Place in about 25ml Buffer 2 and add 5 m L anti-digoxigen-AP conjugate (this is vial 3 from the AntiDig AP-Conjugate kit. The attempted ratios are 1:1000 to 1:5000. Let incubate at room temperature for 10-30 minutes
5. Pour off.
6. Remove unbound conjugate by washing for about 5 minutes in Buffer 1 (PBS also works)
7. Pour off
8. Remove unbound conjugate by washing for a second time with Buffer 1 for five minutes.
9. Pour off
10. Equilibrate membrane for 2 minutes with Buffer 3.
11. Incubate filter with freshly prepared color-substrate solution (Add 200 m L of NBT/BCIP (vial 4 of kit) to 10 ml of Buffer 3. As well, pre-made NBT/BCIP may be purchased).
12. Development may take anywhere from a few minutes to 16 hours. We usually let it sit overnight.
13. To Stop reaction, was the membrane for 5 minutes with Buffer 4.

Note: At SREL, solutions for Cycle sequencing reside in the -20° 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

1. m M M13 primer* 1.0

(*Forward or Reverse accordingly)

Thermal Cycler Settings:

1 cycle:

94° 3 min.

25 cycles:

96° 5 sec.

50° 5 sec.

60° 4 min.

0. cycle:

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:

Next, I name each of these samples using the following nomenclature:

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:

0. 2-5 6-8 )

Again,

Column 1 is the location of the locus on the 96-well PCR plate

Column 2-5 is the meaningful locus name

Column 6-8 is the cycle sequencing reaction strip tube ID

Thus, much information is consolidated. When I fill out the sample sheet on the automated sequencer, I will use the strip tube ID name, and rename each file once I have imported it into the Sequencher software.

- For Big Dye sequencing reactions add the following to each 10m L sequencing reaction:

40m L of 76% ethanol

- Let sit for 15 min. (up to 24 hours)

• Spin in Eppendorf centrifuge (located at USC) at 1500G for 45 min.
• Remove caps (keep clean)
• Put on block lid
• Flip
• Spin at 500 G for 1 minute

• Prepare samples for sequencer leading by adding 40 m L of dye mix (dye + formamid) to each sample.

This protocol makes 50ml of 4.5% 29:1 plyacrylamide gel, 6 M urea (for 36-cm WTR runs)

Mix:

Urea 18 g

Bead ion exchange resin 0.5 g

H2O 20 ml

40% acrylamide stock 5.625 ml

(mix the above together in a hot water bath)

Set up filter:

5X TBE 10 ml

Immediately prior to pouring gel add:

10% APS 250 m L

TEMED 30 m L

- Clean glass well (rinse last with ethanol)

• Wet inserts, flip
• Place inserts on notched glass
• Place "top" glass on notched glass
• Constantly check bottom to ensure that both plates are flush
• Carefully place plates in cartridge with handle up, lift plates over botton bar, ensure hooks flush with notch. Clip in
• Attach plexyglass upper bracket.
• Attach plexyglass bottom bracket. Flip handles slowly at same time. Suck H2O from hole with syringe.
• Avoid bubbles when pouring polyacrylamide through bottom spout. Depress plunger quick and steady.
• Place comb, reverse.
• Wipe away any bubble between comb and plate.
• Insert comb between plates.
• Let gel dry (approximately 1 hour).

• Travis Glenn has developed a computer program for detecting repeats in log files provided by the sequencer.
• It is also effective to visually inspect each sequence for repeats with Sequencher software
• When searching for microsatellite regions, less than the following number of repeats are most likely to not be variable:
• Dinucleotide repeats 10+
• Trinucleotide repeats 7+
• Tetranucleotide repeats 4+

Notes on using Sequencher:

1. Import sequences into sequencher

File/ Import/ Import folder of sequences

2. Save "project"

Save Project as/ "XXX"

3. Trim sequences

This can be done by hand or by command "Trim ends". However, sequences are often poor following a repeat rich region, so mindlessly trimming ends may delete potential target areas. What is most important is to remove sequence associated with the plasmid. Primers designed with sequence included in the plasmid obviously are unlikely to work. The "Find" command can be used to target the GATC sequence associated with the PuC18 vector. Ideally you want about 500bp of good sequence.

4. To combine forward and reverse reactions into a "contig"

Can adjust "Assembly Parameters"/Assemble Automatically

5. Edit the sequence
6. To export the sequence for primer design in Oligo

• If a sequence contains a repeat rich region of suitable size, then sequence the reverse reaction.

Oligo software is used to design primers.

1. Open file

File/ Open (Do not "Import". This feature causes problems.)

2. Scan sequence for microsatellite region

Upon opening a file, you will see two graphs. On the bottom graph, straight lines represent sequences of repeat rich regions.

3. Design primer.

The optimal microsatellite primer design is a segment of DNA sandwiched between the primers 200 to 300 bp in length. This segment includes the repeat plus 10-20 bp of sequence on both sides of the repeat. Primers themselves are optimally 20bp in length. The upper and lower primers do not have to be identical in size.

4. Specify oligo length

Change/ current oligo length (start at 20)

The ideal primer length is 16-24 bp

5. Search for primers

Search/ primer & probes/

PCR Primers/ X Method +/-

Search parameters/ Set (+) range 1 to 60 / Set (—) range 150 to 262 / Default for rest

"Go"

If no primers found, play with parameters.

6. Evaluate primers

Located primers will be described as U (upper) and L (lower).

7. Analyze primers (press "A")

8. It is not possible to save a primer analysis in Oligo, so all primers and analysis should be copied into

a Word document.

The Eppendorf MasterCycler Gradient is ideal for optimizing primers. Contact Brinkmann Instruments, Inc. 1-800-645-3050 for more details.

1. Have someone (Daniel) demonstrate set up.

Use alignment slot to assemble glass plates and spacers. Use water to check for leaks.

2. Can use a big syringe to load polyacrylamide.
3. Use premade 8% polyacrylamide to save on time.
4. Apporximately use 10ml of polyacrylamide stock per minigel. Add 100 m L APS. Add 20 m L TEMED.

8% gel:

25mL 200mL stock 10mL /minigel

40% polyacrylamide 5 mL 40 mL stock

H2O 13 mL 104 mL stock

5X TBE 5 mL 40 mL stock

APS 150 m L nada 100m L

TEMED 30 m L nada 20 m L

 

5. Let polymerize. Recommendation is 45 minutes.
6. Add buffer to buffer tray
7. Run (Biorad Powerpak)

Recommendation is 200V for 45 minutes

I have been trying 100V for 2 hours

8. I have been adding @ 10 m L PCR product per well (same for 100bp standard)

To resolve tetramer repeats, 100-300 bp.

Use 1X TBE

2% or 3% gel (3% gives finer resolution)

Pre-chill buffer.

Stir TBE with stirband and add metaphor to avoid clumps

Mix well before heating in microwave.

To microwave:

Use seran wrap.

Heat 20-30 seconds and swirl

Repeat until dissolved.

Pour.

Gel should be 3-5 mm thick.

Buffer overlay should be 3-5 mm deep.

After gel solidifies at room temperature, chill for 30 minutes at 4° C for best resolution.

Run 4 — 10 V / cm (7 V/cm is ideal)

Cm is measured as the distance between electrodes of the gel box.

It is best to stain after running the gel.

Here it is important to take into account which primers you will want to co-load onto a single well of the automated sequencer. Also, it is recommended to avoid ordering primers labeled with the dye used in your internal standard (this has happened, though not by me).

Although primers may react slightly different when labeled, my experience with reoptimization was relatively painless.

-Time involvement depends on previous experience

Perkin Elmer has an excellent web site which describes Plus A and Stutter. Overloading PCR product on the automated sequencer will result in "pull up" in which false peaks are created on other co-loaded samples and standard due to extreme signals.

-Time investment depends on sample size and technique.

-Time investment depends on sample size and assorted DNA/PCR "good karma".

"Short" polyacrylamide gels may be used to analyze microsatellites. Run time for these is about an hour. As well, the same gel may be reused 2 or 3 times (Lisa Davis, personal communication). GeneScan and Genotyper are the software packages associated with the ABI 377 for microsatellite analysis. Much of the microsatellite analysis process can be automated by creating catagories for allele bins and directly exporting data into a spreadsheet.

My experience with statistical analysis thus far is quite minimal. There are a fair number of computer programs to conduct statistical tests.