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Microsatellite Protocol

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Preparation of reaction mixture 

1. Dilute primers to 10 µM by adding 200 µl 1x TE buffer (see Table 8-9 ). Each primer in the Rice Map Pairs set from Research Genetics is supplied at 200 µl of 20 µM in 1x TE buffer (pH 8).

2. Aliquot 5 µl of the genomic DNA (1.25 ng/µl) in each of the 40 properly labeled 0.5 ml PCR tubes or plate wells.

3. Prepare the cocktail for 40 reactions in 1.5 ml microtube (see Table 8-9) for preparation of MgCl2 and dNTP mix).

Component

Stock

Volume

Final

Volume/40

 

Concentration

(µl)

Concentration

rxn (µl)

supH2O

 

14.1

 

564

PCR buffer

10x

2.5

1x

100

MgCl2

15 mM

1.7

1.0 mM

68

dNTP mix

5 mM

0.5

0.1 mM

20

Primer-reverse

10 µM

0.5

0.2 µM

20

Primer-forward

10 µM

0.5

0.2 µM

20

Commercial Taq

5u/µl

0.2

1 u

8

 Polymerase

 

 

 

 

Note: Homemade Taq Polymerase of 1 unit final concentration can be used. Mix the cocktail by flicking the tube and spin down to collect the mixture.

4. Add 20 µl of cocktail to the genomic DNA and mix gently by flicking the tube.

5. Overlay the mixture with 1 drop (10 µl) mineral oil. Spin down to collect the mixture (for tubes only).

10x PCR buffer

Stock Concentration

Final Concentration

Amount

Tris-HCl, 1 M

100 mM

2.5 ml

KCL, 2 M

500 mM

6.25 ml

Gelatine, 2%

 0.1%

1.25 ml

Amplification 

1. Place tubes/plate in a thermal cycler and allow amplification to proceed with the following temperature profile:

Temperature (oC)

Time (min)

No. of cycles

94

5

1

94

1

35

55

1

72

2

72

5

1

Hold temperature: 4 °

 

2. After amplification remove tubes/plate from thermal cycler and add 12.5 µl 3x STR loading buffer (see Table 8-9). Store at 4 ° for further use.

Note: PCR conditions for microsatellite markers will vary depending on the individual PCR machine and the actual primer used. Annealing and denaturation temperatures and MgCl2 should be adjusted in order to obtain amplified products.

Assembling the glass plate sandwich

(Adapted from Sequi-Gen® GT Nucleic Acid Electrophoresis Cell – Instructional Manual.)

  1. Thoroughly clean both Sequi-Gen® GT integral plate chamber (IPC) glass plate and outer glass plate with liquid soap. Rinse plates with dH2O. Always wear gloves while handling the glass plates. Fingerprints will cause bubbles to form during gel casting.

  2. Place the outer glass plate flat on a lab table. Wipe the entire surface of the outer glass plate with fresh binding solution (see Table 8-9). Spread evenly with Kimwipe® tissue. Allow to dry for 5 min.

  3. Place the IPC flat on the lab table with the glass plate facing upward. Apply 1 ml of Sigmacote®. Spread evenly over the entire surface of the glass plate with Kimwipe® tissue. Allow to dry.

  4. Position one 0.4 mm side spacer along each edge of the IPC glass plate. The bottom edges of the spacer and the IPC glass plate should be flush and the long edge of the spacer should be next to the plastic lip of the IPC panel.

  5. Place the front of the outer plate on to the IPC and spacers with the coated surface facing down.

  6. With both hands, stand the IPC/outer glass plate sandwich on the lab table with the outer glass plate facing away from you.

  7. Slide the clamps over the sides of the IPC assembly. The lever of the clamp should be on the IPC panel side of the assembly and facing away from the unit and perpendicular to the IPC panel for the clamps to slide easily onto the assembly. Secure the clamps to the IPC/outer glass plate sandwich by moving the levers towards the IPC panel.

  8. Place the assembly in a flat surface with the IPC panel facing up. Check if the glass plates and the spacers are flush.

  9. Place the precision caster base on the lab table with the open cavity facing up. Place the gray precision gasket into the base. The cam pegs in the precision caster must be pulled out to accommodate the apparatus.

  10. Place bottom edge of the IPC assembly into the precision caster base with the bottom edge of the assembly resting against the gray gasket of the precision caster base.

  11. Once resting on the gasket, use the cam pegs to connect the base to the clamps. Push each cam peg into the corresponding hole on the clamp with the lever in the up position. Slight downward pressure applied to the top of the IPC assemble may be required to engage each cam peg.

  12. Lay the IPC assembly flat on the lab table with the drain port facing up and the precision caster base facing towards you. Make sure that the assembly is level to prevent gel leakage.

Gel preparation

  1. Prepare the gel by mixing 100 ml 6% acrylamide solution, 50 µl TEMED, and 600 µl 10% APS. Mix gently by swirling.

  2. Fill a 100 ml syringe slowly with the solution and attach the tubing assembly. Remove the bubbles from the syringe by forcing some of the gel out.

  3. When all the bubbles are removed from the tubing, place the luer taper into the injection port of the precision caster base. Slowly inject the gel solution on to the glass plate sandwich. Do not remove the tubing until the gel has polymerized.

  4. Insert the flat edge of a sharktooth comb 5 mm past the edge of the outer glass plate. Clamp it with a large metal binder to hold it in place.

  5. Let the gel polymerize for 30-60 min. Remove the tubing and the precision caster base from the assembly. Clean the caster base and gasket of polymerized gel solution with tap H2O, followed by a dH2O rinse.

Pre- electrophoresis

  1. Fill the lower buffer chamber with 350-500 ml 1x TBE buffer (see Table 8-9).

  2. Gently lower the gel assembly to the universal base. Insert the stabilizer bar.

  3. Fill the upper buffer chamber with 1400 ml 1x TBE buffer. The level of the buffer should be about 1 cm from the top of the fill spout at all times during the run. Gel electrophoresis buffer can be heated to 50 ° in a microwave oven before adding buffer into the upper buffer chamber. This will reduced the time needed to bring the gel to the appropriate run temperature before sample loading, and will greatly reduced pre-electrophoresis run time.

  4. Remove the comb from between the glass plates. Clean the well area using a syringe. Make sure to remove air bubbles and unpolymerized acrylamide.

  5. Adhere a gel temperature indicator onto the outside of the outer glass plate, somewhere near the center to monitor the gel temperature during electrophoresis.

  6. Attach the top and bottom safety covers. Attach electrode wires to the power supply and pre-run the gel at 120 W to achieve a gel surface temperature of approximately 45-50 ° Pre-electrophoresis prior to sample loading will create a uniform gel temperature and bring the gel temperature to the recommended run temperature. This will help eliminate any smile pattern from developing early in the run.

Loading the DNA samples and gel electrophoresis

  1. Denature DNA samples by heating in a thermal cycler at 95 ° for 5 min and immediate chilling on ice.

  2. After the pre-run turn off the power supply and remove the top safety cover. Clean the well area again.

  3. Carefully insert the teeth of the sharktooth comb into the gel .5-1 mm deep.

  4. Load 6 µl for 46 wells or 4 µl for 72 wells of each sample into the wells. Loading of samples should not exceed 20 min to prevent cooling of the gel and to maintain the denatured state of DNA.

  5. Attach top safety cover. Turn on power supply and run the gel at 120 W maintaining the tempearture at 50 ° for 1 h. Running time would vary depending upon the primer used. Generally, stop the run after the bromphenol blue (leading dye) reaches the bottom of the gel.

Disassembly

  1. After electrophoresis, turn off the power supply and remove both safety covers.

  2. The upper buffer chamber can be partially emptied by inserting the drain port connector into the drain port on the IPC. Buffer should drain immediately from the IPC.

  3. After the upper chamber is emptied to the level of the drain port, pull out the stabilizer bar and remove the IPC assembly. Blot the bottom edge of the IPC assembly onto absorbent paper.

  4. Carefully pour the remaining upper buffer out of the IPC assembly into a container. Also, carefully drain the lower buffer from the universal base into a container. Never store buffers in an IPC. Never add buffer to an IPC unless the clamps are in place.

  5. Remove the clamps from the IPC assembly by first pulling the levers away from the IPC and then sliding the clamps off the IPC assembly.

  6. Lay the IPC assembly flat on a lab table with the outer glass plate facing up. Carefully separate the glass plate by pulling up gently near the top of the outer plate. The gel should come apart from the IPC and become strongly affixed to the outer glass plate. Remove the comb and side spacers.

Staining

(Adapted from Promega’s Silver Sequence® DNA Sequencing System Technical Manual Rev. 8/96)

  1. Place outer glass plate with the gel in a plexiglass tray with the fix/stop solution (see Table 8-9) for 20 min with continuous shaking. Do not discard the fix/stop solution. Do this step inside a fume hood.

  2. Wash the gel thrice for 2 min each in a tray with upH2O with continuous shaking.

  3. Stain the gel with silver stain solution (see Table 8-9) for 30 min in a tray with continuous shaking.

  4. Rinse the gel in a tray with upH2O for 10 sec.

  5. Transfer the gel to a tray with developer solution (pre cooled to 4-10 ° ) (see Table 8-9) for 2-5 min (or as soon as the bands appear) with shaking.

  6. Return the gel to the fix/stop solution for 5-6 min.

  7. Rinse the gel in a tray with upH2O for 2-3 min and allow the gel to dry at room temperature or at 50 °

Documentation

  1. In a dark room with a red safelight place the gel on a light box. The light box should have a white bulb.

  2. Position the Promega APC film with the emulsion side down over the gel.

  3. Turn on the lightbox and expose the film for 10 sec. Film exposure may vary with different lightboxes or with different batches of APC Film. Make test exposures first by exposing small strips of film at varying times.

  4. Develop the film in the following solutions (see Table 8-9 for developer and fixer solutions):

    Time

    Solution

    1-3 min*

    Kodak® GBX Developer

    1 min

    distilled water

    3 min

    Kodak® GBX Fixer

    1 min

    distilled water

    *will vary depending on exposure conditions.

  5. Air dry the APC film. This is now ready for scoring.