This protocol is an amalgamation of protocols used and provided by Vos et al., Brent Barrett (WSU), Greg Penner (Ag CA), A.S. Reddy (Texas A&M), Gibco BRL, and others as well as additions and changes made in Wheat lab.

I. Restriction Digestion of Genomic DNA
 

	Starting [ ]	Final [ ]	Per rxn (µl)
OnePhorAll (OPA, Pharmacia)	10X	1X	5.0
Mse I (New England Biolabs)	4U/µl	5U	1.25
Eco RI (GibcoBRL)or Pst I (Promega)	10U/µl	5U	0.5
BSA (comes w/ Mse I)	10µg/µl	5µg	0.5
ddH2O			32.75
Genomic DNA	50-250ng/µl		10µl
Total			50µl

 

1. Heat one oven to 70⁰ and the other to 37⁰.
2. Distribute 40µl of cocktail in each labeled tube.
3. Add 10µl of DNA to each tube.
4. Vortex and briefly centrifuge.
5. Incubate @ 37⁰ for 3 hours. Agitate every hour or so. I do a quick vortex so the sample stays in bottom of tube and centrifugation is not needed.
6. Inactivate enzyme @ 70⁰ for 15 min.

II. Adapter preparation

Complete during or before digestion

Eco RI Adapter (120 ligation recipe)
 

Eco RI.1 oligo (1 µg/µl)	3.4µl
Eco RI.2 oligo (1 µg/µl)	3.0µl
OPA	6.0µl
ddH2O	107.6µl

 

Mse I Adapter (120 ligation recipe)*
 

Mse I.1 oligo (0.5 µg/µl)	64.0µl
Mse I.2 oligo (0.5 µg/µl)	56.0µl
OPA	7.0µl

 

*Mse I oligos may need to be speed vacuumed in order to increase concentration.

Mix in thermocycler tubes and run file #44.

65⁰C for 10 min.

37⁰C for 10 min.

25⁰C for 10 min.

Store at -20⁰C.

III. Ligation of Adapters.

 
 

	Per rxn
Eco RI adapter	1µl
Mse I adapter	1µl
T4 DNA ligase 10X buffer	1µl
T4 DNA ligase (3U/µl, Promega)	0.33µl
ddH2O	6.7µl

 

1. Add 10 ul of ligation mix to 50 ul of digested DNA. Vortex and briefly centrifuge.
2. Incubate at room temperature for 3 hrs. Agitate every hour or so as above.

IV. Pre amplification Reactions

 
 

Eco RI + A oligo (50ng/µl)	0.5µl
Mse I + C oligo (50ng/µl)	0.5µl
dNTPs (5mM, Gibco as 100mM)	2µl
10X PCR buffer (w/ Taq)	2µl
Taq polymerase (5U/µl ,Promega)	0.1µl
MgCl2 (w/ Taq)	1.2µl
ddH2O	11.9µl
Template DNA from restriction/ligation	2µl

 

Thermocycler file #36
 

94	2 min
94	1 min	26 cycles
56	1 min.
72	1 min.
72	5 min.
4	hold

 

1. Transfer PCR product into new tubes with 100µl sterile ddH₂O.
2. Blot testing can test reaction success. Dot 2µl of Ethidium bromide (2µg/µl) and 3µl of product on plexi-glass. Use 3µl of cocktail as control. Visualize dots using UV box.

 

V. Selective amplification
 

		24	36	48
Eco RI + ANN oligo (50ng/µl)	0.5µl	13	19.5	26
Mse I + CNN oligo (50ng/µl)	0.6µl	15.6	23.4	31.2
dNTPs (5mM, Gibco as 100mM)	0.8µl	20.8	31.2	41.6
10X PCR buffer (w/ Taq)	2.0µl	52	78	1.4
Taq polymerase (5U/µl ,Promega)	0.08µl	2.1	3.12	4.16
MgCl2 (w/ Taq)	1.2µl	31.2	46.8	62.4
ddH2O	13.82µl	360	540	720
Dilute template DNA from pre-selective PCR	1µl

 

Thermocycler file #20
 

94	2 min
94	30s	12 cycles, decrease annealing temp by 0.7 each
65	30s
72	1 min.
94	30s	23 cycles
56	30s
72	1 min.
72	2 min.
4	hold

 

3. Test product using dot blot if necessary.
4. Combine 8µl formamide-loading buffer and PCR product.

VI. Gel electrophoresis

1. Acrylamide gel solution

42g Urea

10ml 10x TBE

15ml 40% acrylamide

water up to 100mls

 

2. Combine urea, TBE, and approx. 25ml water in a beaker. Stir with heat until urea dissolves.
3. Transfer solution to 100ml-graduated cylinder and add water up to 85ml. Transfer to vacuum flask.
4. Add acrylamide to flask and degas for approx. 10min.
5. Transfer solution to beaker and add 100µl TEMED and 500µl 10% fresh APS. Draw solution into syringe. Keep tip submerged at all times.
6. Place tube on syringe and turn it upward. Push air out of tube and pinch end of tube. Insert into Caster base.

 

7. Glass preparation (all glass must be scrupulously clean!)
8. Wipe IPC unit with chem-wipe and ethanol.
9. Glass should be treated with Sigmacote about every 5 gels run or until top of gel sticks to long glass. Saturate a chem-wipe with Sigmacote and wipe vertically and horizontally. Wait five minutes and wipe glass three times with ethanol. Change gloves.
10. Wipe long glass with ethanol and chem-wipe.
11. In an Eppindorf tube combine 1ml of 95%EtOH 0.05%Acetic acid and 2µl of bind silane.
12. Treat glass same as above description of Sigmacote. Use a great deal of pressure when wiping with EtOH. Change gloves.
13. In the event of a contamination of either Sigmacote or Bind silane on the respective glass, soak in 10%NaOH.
14. While horizontal, place spacers on IPC and long glass on top.
15. Erect vertically and clamp side braces.
16. Attach Caster base insert pegs and turn. Be sure to do this while vertical and that you can see the space in between glass plates through Caster base hole.
17. Check to see if comb will easily insert between glass. If not, adjust.
18. Lean clamps on top of tube racks.
19. Inject gel solution.
20. Insert combs and adjust unit to horizontal position.
21. Allow gel to polymerize for at least 1 hour.

VII. Gel loading

1. Fill bottom tray with 1X TBE so about ½ inch of the bottom of gel unit is submerged. Fill IPC until ½ inch above short glass. Use needle and flush out well
2. Run gel at 75W for 1hr.
3. Flush well again and insert comb without piercing gel.
4. Load 4.5µl sample.
5. Run gel for 10min and then remove comb (good time to make fix/stop and developing solution).
6. Run gel for total of 2hr and 50min. (Light blue dye should migrate 1 inch below bottom rib of IPC.
7. Insert tube in IPC and drain buffer.
8. Pull glass apart and wash IPC.

VIII Silver Staining

From Promega Technical Manual and Sam and Suzanne Downey empirical knowledge.

1. Separate plates while keeping the gel attached to short glass.

 

2. Fix the gel: Place gel in tray, cover with cold fix/stop solution and agitate well for 20 minutes. Gel may be stored in fix/stop solution overnight. Save fix/stop solution and place back in freezer.

 

3. Wash the gel: Rinse the gel 3 times for 2-3 min. each in ddH₂O using agitation. Lift gel from solution and allow to drain 10-20 seconds.

 

4. Stain the gel: Transfer the gel to staining solution and agitate well for 30 minutes.

 

5. Pour 1L of the developing solution into a tray. Transfer staining solution to beaker. Rinse tray and fill with ddH₂O.

 

6. Rinse gel for 5-10 seconds ONLY. Transfer to developing solution.

 

7. Agitate in developing solution until bands begin to appear. Transfer gel to remaining chilled developing solution for 2-3 minutes.

 

8. Fix the gel: add 1L of Fix/stop solution directly to developing solution and agitate for 2-3 minutes

 

9. Rinse gel twice for two minutes each in ddH₂O.

 

10. Dry gel on glass

Fix/stop solution Staining solution

200ml of glacial acidic acid 2g (1 packet) of silver nitrate (AgNO₃)

1,800ml ultrapure water 3ml (1 vial) of 37% Formaldehyde

Freeze for approx. 3 hours 2L ultrapure water

Developing solution

60g (1 packet) Sodium Carbonate (Na₂CO₃)

2L ultrapure water

**chill to 10^oC. I place sol. In freezer for approx 4 hours and stir to break up ice prior to use.

Immediately before use add

3ml (1 vial) of 37% Formaldehyde

400m l aliquot Sodium Thiosulfate (discard remaining)

IX. Gel scoring and scanning.

1. Scan gel in two sections without two options selected. Save as compressed tif and jpg.
2. Score gel while still on glass and make notes on printout of scanned image.
3. Keep original score sheet and gel printout in folder
4. Record gel in record form with all pertinent details.

X. Gel preservation

1. Soak gel in 3% NaOH with gentle agitation for 30 to 60min, or until edge of corner of the gel starts coming loose. If gel does not come loose, tease a corner and pull gently. If it peels easily, gel is ready for transfer. Loosen edges with razor blade to facilitate transfer.
2. Carefully transfer gel to 3.5% acetic acid and soak for 3 min without agitation. Rinse in ddH₂O for 2 minutes without agitation.
3. Drain excess water from gel and smooth a sheet of chromatography paper over gel.
4. Very slowly pull edge or corner up while gel adheres to paper. Use a razor blade to persuade any lagging parts of the gel.
5. Cover gel with plastic wrap and dry on gel dryer at 70^o for 2 hrs.

Oligo Fragments

 
 

EcoRI Linker 1	CTC GTA GAC TGC GTA CC
EcoRI Linker 2	AAT TGG TAC GCA GTC TAC
EcoRI +A	GAC TGC GTA CCA ATT CA
Pst I Linker 1	CTC GTA GAC TGC GTA CAT GCA
Pst I Linker 2	TGT ACG CAG TCT AC
Pst I +A	GAC TGC GTA CAT GCA GAC A
Mse I Linker 1	GAC GAT GAG TCC TGA G
Mse I Linker 1	TAC TCA GGA CTC AT
Mse I +C	GAT GAG TCC TGA GTA AC